Anti-ICOS antibodies

ABSTRACT

Antibodies that bind ICOS (Inducible T cell Co-Stimulator). Therapeutic use of anti-ICOS antibodies for modulating the ratio between regulatory T cells and effector T cells, to stimulate the immune system of patients, including use in treating cancers. Methods of producing anti-ICOS antibodies, including species cross-reactive antibodies, using transgenic knock-out mice.

FIELD OF THE INVENTION

This invention relates to compositions for stimulating the mammalianimmune response, especially the T cell response. The invention alsorelates to medical use of such compositions in immuno-oncology,including anti-tumour therapy by promotion of anti-tumour T cellresponse in a patient, as well as to use of the compositions in otherdiseases and conditions where it is of therapeutic benefit to modulatethe balance between effector T cells and regulatory T cells in favour ofeffector T cell activity, for example through stimulation of effector Tcells and/or through depletion of regulatory T cells.

BACKGROUND

ICOS (Inducible T cell Co-Stimulator) is a member of the CD28 genefamily involved in regulating immune responses, in particular humoralimmune responses, first identified in 1999 [1]. It is a 55 kDatransmembrane protein, existing as a disulphide linked homodimer withtwo differentially glycosylated subunits. ICOS is exclusively expressedon T lymphocytes, and is found on a variety of T cell subsets. It ispresent at low levels on naïve T lymphocytes but its expression israpidly induced upon immune activation, being upregulated in response topro-inflammatory stimuli such as on engagement of TCR and co-stimulationwith CD28 [2, 3]. ICOS plays a role in the late phase of T cellactivation, memory T cell formation and importantly in the regulation ofhumoral responses through T cell dependent B cell responses [4, 5].Intracellularly, ICOS binds PI3K and activates the kinasesphophoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB).Activation of ICOS prevents cell death and upregulates cellularmetabolism. In the absence of ICOS (ICOS knock-out) or in the presenceof anti-ICOS neutralising antibodies there would be a suppression ofpro-inflammatory responses.

ICOS binds to ICOS ligand (ICOSL) expressed on B-cells and antigenpresenting cells (APC) [6, 7]. As a co-stimulatory molecule it serves toregulate TCR mediated immune responses and antibody responses toantigen. The expression of ICOS on T regulatory cells may be important,as it has been suggested that this cell type plays a negative role inimmunosurveillance of cancer cells—there is emerging evidence for thisin ovarian cancer [8]. Importantly, ICOS expression has been reported tobe higher on intratumoural regulatory T cells (TRegs) compared with CD4+and CD8+ effector cells that are present in the tumour microenvironment.Depletion of TRegs using antibodies with Fc-mediated cellular effectorfunction has demonstrated strong anti-tumour efficacy in a pre-clinicalmodel [9]. Mounting evidence implicates ICOS in an anti-tumour effect inboth animal models as well as patients treated with immune-checkpointinhibitors. In mice deficient in ICOS or ICOSL the anti-tumor effect ofanti-CTLA4 therapy is diminished [10] while in normal mice ICOS ligandincreases the effectiveness of anti-CTLA4 treatment in melanoma andprostate cancer [11]. Furthermore, in humans a retrospective study ofadvanced melanoma patients showed increased levels of ICOS followingipilimumab (anti-CTLA4) treatment [12]. In addition, ICOS expression isupregulated in bladder cancer patients treated with anti-CTLA4 [13]. Ithas also been observed that in cancer patients treated with anti-CTLA4therapy the bulk of tumour specific IFNγ producing CD4 T-cells are ICOSpositive while sustained elevation of ICOS positive CD4 T cellscorrelates with survival [12, 13, 14].

WO2016/120789 described anti-ICOS antibodies and proposed their use foractivating T cells and for treating cancer, infectious disease and/orsepsis. A number of murine anti-ICOS antibodies were generated, of whicha sub-set were reported to be agonists of the human ICOS receptor. Theantibody “422.2” was selected as the lead anti-ICOS antibody and washumanised to produce a human “IgG4PE” antibody designated “H2L5”. H2L5was reported to have an affinity of 1.34 nM for human ICOS and 0.95 nMfor cynomolgus ICOS, to induce cytokine production in T cells, and toupregulate T cell activation markers in conjunction with CD3stimulation. However, mice bearing implanted human melanoma cells werereported to show only minimal tumour growth delay or increase insurvival when treated with H2L5 hIgG4PE, compared with control treatedgroup. The antibody also failed to produce significant furtherinhibition of tumour growth in combination experiments with ipilimumab(anti-CTLA-4) or pembrolizumab (anti-PD-1), compared with ipilimumab orpembrolizumab monotherapy. Finally, In mice bearing implanted coloncancer cells (CT26), low doses of a mouse cross reactive surrogate ofH2L5 in combination with a mouse surrogate of ipilimumab orpembrolizumab only mildly improved overall survival compared withanti-CTL4 and anti-PD1 therapy alone. A similar lack of strongtherapeutic benefit was shown in mice bearing implanted EMT6 cells.

WO2016/154177 described further examples of anti-ICOS antibodies. Theseantibodies were reported to be agonists of CD4+ T cells, includingeffector CD8+ T cells (TEff), and to deplete T regulator cells (TRegs).Selective effects of the antibodies on TEff vs TReg cells weredescribed, whereby the antibodies could preferentially deplete TRegswhile having minimal effect on TEffs that express a lower level of ICOS.The anti-ICOS antibodies were proposed for use in treating cancer, andcombination therapy with anti-PD-1 or anti-PD-L1 antibodies wasdescribed.

SUMMARY OF THE INVENTION

An antibody to ICOS that acts to increase effector T cell activityrepresents a therapeutic approach in immunooncology and in other medicalcontexts where a CD8+ T cell response is beneficial, including variousdiseases and conditions and in vaccination regimens. In many diseasesand conditions involving an immune component, a balance exists betweeneffector T cells (TEff) which exert the CD8+ T cell immune response, andregulatory T cells (TReg) which suppress that immune response bydownregulating TEffs. The present invention relates to antibodies thatmodulate this TEff/TReg balance in favour of effector T cell activity.Antibodies that trigger the depletion of ICOS highly positive regulatoryT cells would relieve the suppression of TEffs, and thus have a neteffect of promoting the effector T cell response. An additional orcomplementary mechanism for an anti-ICOS antibody is via agonisticactivity at the ICOS receptor level, to stimulate the effector T cellresponse.

The relative expression of ICOS on effector T cells (TEff) compared withregulatory T cells (TReg), and the relative activities of these cellpopulations, will influence the overall effect of an anti-ICOS antibodyin vivo. An envisaged mode of action combines agonism of effector Tcells with depletion of ICOS positive regulatory T cells. Differentialand even opposing effects on these two different T cell populations maybe achievable due to their different levels of ICOS expression.Dual-engineering of the variable and constant regions respectively of ananti-ICOS antibody can provide a molecule that exerts a net positiveeffect on effector T cell response by affecting the CD8/TReg ratio. Anantigen-binding domain of an agonist antibody, which activates the ICOSreceptor, may be combined with an antibody constant (Fc) region thatpromotes downregulation and/or clearance of highly expressing cells towhich the antibody is bound. An effector positive constant region may beused to recruit cellular effector functions against the target cells(TRegs), e.g., to promote antibody-dependent cell-mediated cytotoxicity(ADCC) or antibody dependent cell phagocytosis (ADCP). The antibody maythus act both to promote effector T cell activation and to downregulateimmunosuppressive T Regulatory cells. Since ICOS is more highlyexpressed on TRegs than on TEffs, a therapeutic balance may be achievedwhereby Teff function is promoted while TRegs are depleted, resulting ina net increase in the T cell immune response (e.g, anti-tumour responseor other therapeutically beneficial T cell response).

Several pre-clinical and clinical studies have shown a strong positivecorrelation between high effector T-cell to T-reg cell ratio in thetumour microenvironment (TME) and overall survival. In ovarian cancerpatients the ratio of CD8:T-reg cells has been reported to be anindicator of good clinical outcome [15]. A similar observation was madein metastatic melanoma patients after receiving ipilumumab [16]. Inpre-clinical studies, it has also been shown that high effectorcell:T-reg ratio in TME is associated with anti-tumour response [43].

This invention provides antibodies that bind human ICOS. The antibodiestarget the ICOS extracellular domain and thereby bind to T cellsexpressing ICOS. Examples are provided of antibodies that have beendesigned to have an agonistic effect on ICOS, thus enhancing thefunction of effector T cells, as indicated by an ability to increaseIFNγ expression and secretion. As noted, anti-ICOS antibodies may alsobe engineered to deplete cells to which they bind, which should have theeffect of preferentially downregulating regulatory T cells, lifting thesuppressive effect of these cells on the effector T cell response andthus promoting the effector T cell response overall. Regardless of theirmechanism of action, it is demonstrated empirically that anti-ICOSantibodies according to the present invention do stimulate T cellresponse and have anti-tumour effects in vivo, as shown in the Examples.Through selection of appropriate antibody formats such as thoseincluding constant regions with a desired level of Fc effector function,or absence of such effector function where appropriate, the anti-ICOSantibodies may be tailored for use in a variety of medical contextsincluding treatment of diseases and conditions in which an effector Tcell response is beneficial and/or where suppression of regulatory Tcells is desired.

Exemplary antibodies include STIM001, STIM002, STIM002-B, STIM003,STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, the sequencesof which are set out herein.

An antibody according to the invention may be one that competes forbinding to human ICOS with an antibody (e.g., human IgG1, or an scFv)comprising the heavy and light chain complementarity determining regions(CDRs) of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 or STIM009, optionally an antibody comprisingthe VH and VL domains of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009.

An antibody according to the present invention may comprise one or moreCDRs of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 and STIM009 (e.g., all 6 CDRs of any suchantibody, or a set of HCDRs and/or LCDRs) or variants thereof asdescribed herein.

The antibody may comprise an antibody VH domain comprising CDRs HCDR1,HCDR2 and HCDR3 and an antibody VL domain comprising CDRs LCDR1, LCDR2and LCDR3, wherein the HCDR3 is an HCDR3 of an antibody selected fromSTIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006,STIM007, STIM008 and STIM009 or comprises that HCDR3 with 1, 2, 3, 4 or5 amino acid alterations. The HCDR2 may be the HCDR2 of the selectedantibody or it may comprise that HCDR2 with 1, 2, 3, 4 or 5 amino acidalterations. The HCDR1 may be the HCDR1 of the selected antibody or itmay comprise that HCDR1 with 1, 2, 3, 4 or 5 amino acid alterations.

The antibody may comprise an antibody VL domain comprising CDRs HCDR1,HCDR2 and HCDR3 and an antibody VL domain comprising CDRs LCDR1, LCDR2and LCDR3, wherein the LCDR3 is an LCDR3 of an antibody selected fromSTIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006,STIM007, STIM008 and STIM009 or comprises that LCDR3 with 1, 2, 3, 4 or5 amino acid alterations. The LCDR2 may be the LCDR2 of the selectedantibody or it may comprise that LCDR2 with 1, 2, 3, 4 or 5 amino acidalterations. The LCDR1 may be the LCDR1 of the selected antibody or itmay comprise that LCDR1 with 1, 2, 3, 4 or 5 amino acid alterations.

An antibody may comprise:

an antibody VH domain comprising complementarity determining regionsHCDR1, HCDR2 and HCDR3, and

an antibody VL domain comprising complementarity determining regionsLCDR1, LCDR2 and LCDR3,

wherein the heavy chain complementarity determining regions are those ofSTIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006,STIM007, STIM008 or STIM009 or comprise the STIM001, STIM002, STIM002-B,STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 heavychain complementarity determining regions with 1, 2, 3, 4 or 5 aminoacid alterations; and/or

wherein the light chain complementarity determining regions are those ofantibody STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 or STIM009, or comprise the STIM001, STIM002,STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 orSTIM009 light chain complementarity determining regions with 1, 2, 3, 4or 5 amino acid alterations.

An antibody may comprise a VH domain comprising a set of heavy chaincomplementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3,wherein

-   -   HCDR1 is the HCDR1 of STIM003,    -   HCDR2 is the HCDR2 of STIM003,    -   HCDR3 is the HCDR3 of STIM003,

or comprising that set of HCDRs with 1, 2, 3, 4, 5 or 6 amino acidalterations.

An antibody may comprise a VL domain comprising a set of light chaincomplementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3,wherein

-   -   LCDR1 is the LCDR1 of STIM003,    -   LCDR2 is the LCDR2 of STIM003,    -   LCDR3 is the LCDR3 of STIM003,

or comprising that set of LCDRs with 1, 2, 3 or 4 amino acidalterations.

Amino acid alterations (e.g., substitutions) may be at any residueposition in the CDRs. Examples of amino acid alterations are thoseillustrated in FIG. 35, FIG. 36 and FIG. 37, which show alignments ofvariant sequences of anti-ICOS antibodies. Thus, an amino acidalteration in a STIM003 CDR may be a substitution of the residue presentat the corresponding position in antibody CL-74570 or antibody CL-71642as indicated in FIG. 36.

Example amino acid alterations in STIM003 CDRs are substitutions at thefollowing residue positions, defined according to IMGT:

In HCDR1, substitution at IMGT position 28, optionally a conservativesubstitution, e.g., V28F.

In HCDR2, substitution at IMGT position 59, 63 and/or 64. Optionally thesubstitution at position 59 is N59I, the substitution at position 63 isG63D and/or the substitution at position 64 is D64N and/or D64S.

In HCDR3, substitution at IMGT position 106, 108, 109 and/or 112.Optionally the substitution at position 106 is R106A, the substitutionat position 108 is F108Y, the substitution at position 109 is Y109Fand/or the substitution at position 112 is H112N.

In LCDR1, substitution at position 36, e.g., R36S.

In LCDR3, substitution at position 105, 108 and/or 109. Optionally thesubstitution at position 105 is H105Q, the substitution at position 108is D108G and/or the substitution at position 109 is M109N or M109S.

Antibodies of the invention may comprise VH and/or VL domain frameworkregions corresponding to human germline gene segment sequences. Forexample, it may comprise one or more framework regions of STIM001,STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008or STIM009. The framework region or framework regions may be a FR1, FR2,FR3 and/or FR4.

As described in Example 12, Table E12-1 shows the human germline V, Dand J gene segments that generated the VH domains of these antibodiesthrough recombination and Table E12-2 shows the human germline V and Jgene segments that generated the VL domains of these antibodies throughrecombination. Antibody VH and VL domains of the present invention maybe based on these V(D)J segments.

An antibody of the invention may comprise an antibody VH domain which

(i) is derived from recombination of a human heavy chain V gene segment,a human heavy chain D gene segment and a human heavy chain J genesegment, wherein

the V segment is IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01),IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g., V2-5*10);

the D gene segment is IGHD6-19 (e.g., IGHD6-19*01), IGHD3-10 (e.g.,IGHD3-10*01) or IGHD3-9 (e.g., IGHD3-9*01); and/or

the J gene segment is IGHJ6 (e.g., IGHJ6*02), IGHJ4 (e.g., IGHJ4*02) orIGHJ3 (e.g., IGHJ3*02), or

(ii) comprises framework regions FR1, FR2, FR3 and FR4, wherein

FR1 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01),IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations,

FR2 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01),IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations,

FR3 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01),IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,V2-510), optionally with 1, 2, 3, 4 or 5 amino acid alterations, and/or

FR4 aligns with human germline J gene segment IGJH6 (e.g., JH6*02),IGJH4 (e.g., JH4*02) or IGJH3 (e.g., JH3*02), optionally with 1, 2, 3, 4or 5 amino acid alterations.

FR1, FR2 and FR3 of the VH domain typically align with the same germlineV gene segment. Thus, for example, the antibody may comprise a VH domainderived from recombination of human heavy chain V gene segment IGHV3-20(e.g., VH3-20*d01), a human heavy chain D gene segment and a human heavychain J gene segment IGJH4 (e.g., JH4*02). An antibody may comprise VHdomain framework regions FR1, FR2, FR3 and FR4, wherein FR1, FR2 and FR3each align with human germline V gene segment IGHV3-20 (e.g.,IGVH3-20*d01) with up to 1, 2, 3, 4 or 5 amino acid alterations, and aFR4 that aligns with human germline J gene segment IGHJ4 (e.g.,IGHJ4*02) with up to 1, 2, 3, 4 or 5 amino acid alterations. Alignmentmay be exact, but in some cases one or more residues can be mutated fromgermline, so there may be amino acid substitutions present, or in rarercases deletions or insertions.

An antibody of the invention may comprise an antibody VL domain which

(i) is derived from recombination of a human light chain V gene segmentand a human light chain J gene segment, wherein

the V segment is IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20 (e.g.,IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g.,IGKV3-11*01), and/or

the J gene segment is IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g., IGKJ2*04),IGLJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g., IGKJ1*01); or

(ii) comprises framework regions FR1, FR2, FR3 and FR4, wherein

FR1 aligns with human germline V gene segment IGKV2-28 (e.g.,IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g.,IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3,4 or 5 amino acid alterations,

FR2 aligns with human germline V gene segment IGKV2-28 (e.g.,IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g.,IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3,4 or 5 amino acid alterations,

FR3 aligns with human germline V gene segment IGKV2-28 (e.g.,IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g.,IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3,4 or 5 amino acid alterations, and/or

FR4 aligns with human germline J gene segment IGKJ4 (e.g., IGKJ4*01),IGKJ2 (e.g., IGKJ2*04), IGKJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g.,IGKJ1*01), optionally with 1, 2, 3, 4 or 5 amino acid alterations.

FR1, FR2 and FR3 of the VL domain typically align with the same germlineV gene segment. Thus, for example, the antibody may comprise a VL domainderived from recombination of human light chain V gene segment IGKV3-20(e.g., IGKV3-20*01) and human light chain J gene segment IGKJ3 (e.g.,IGKJ3*01). An antibody may comprise VL domain framework regions FR1,FR2, FR3 and FR4, wherein FR1, FR2 and FR3 each align with humangermline V gene segment IGKV3-20 (e.g., IGKV3-20*01) with up to 1, 2, 3,4 or 5 amino acid alterations, and a FR4 that aligns with human germlineJ gene segment IGKJ3 (e.g., IGKJ3*01) with up to 1, 2, 3, 4 or 5 aminoacid alterations. Alignment may be exact, but in some cases one or moreresidues can be mutated from germline, so there may be amino acidsubstitutions present, or in rarer cases deletions or insertions.

An antibody according to the invention may comprise an antibody VHdomain which is the VH domain of STIM001, STIM002, STIM002-B, STIM003,STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009, or which hasan amino acid sequence at least 90% identical to the antibody VH domainsequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 or STIM009. The amino acid sequence identitymay be at least 95%.

The antibody may comprise an antibody VL domain which is the VL domainof STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006,STIM007, STIM008 or STIM009, or which has an amino acid sequence atleast 90% identical to the antibody VL domain sequence of STIM001,STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008or STIM009. The amino acid sequence identity may be at least 95%.

An antibody VH domain having the HCDRs of STIM001, STIM002, STIM002-B,STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, orhaving a variant of those CDRs, may be paired with an antibody VL domainhaving the LCDRs of the same antibody, or having a variant of thoseCDRs. Similarly, the VH domain of any of STIM001, STIM002, STIM002-B,STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or avariant of that VH domain, may be paired with a VL domain of the sameantibody, or a VL domain variant of the same antibody.

For instance, the antibody may comprise the antibody STIM001 VH domainand the STIM001 VL domain. In another example, the antibody may comprisethe antibody STIM002 VH domain and the STIM002 VL domain. In anotherexample, the antibody may comprise the antibody STIM003 VH domain andthe STIM003 VL domain.

Antibodies may include constant regions, optionally human heavy and/orlight chain constant regions. An exemplary isotype is IgG, e.g., humanIgG1.

Further aspects of the invention include nucleic acid molecules encodingsequences of the antibodies described herein, host cells containing suchnucleic acids, and methods of producing the antibodies by culturing thehost cells and expressing and optionally isolating or purifying theantibodies. The expressed antibody is thereby obtained. VH and VLdomains of antibodies described herein may similarly be produced and areaspects of the present invention. Pharmaceutical compositions comprisingthe antibodies are also provided.

Other aspects of the invention relate to ICOS knock out non-humananimals and their use for generating antibodies to human ICOS. In anICOS knock out animal, ICOS is not expressed, for example because thegene encoding ICOS has been inactivated or deleted from the animal'sgenome. Such animals are useful for generating species cross-reactiveantibodies, which recognise both human ICOS and ICOS from the non-humanspecies. The normal process of immune tolerance means that lymphocytesthat recognise “self” antigens are deleted or inactivated to preventautoimmune reactions in the body, whereas the absence of the endogenousICOS antigen in the non-human knock out animal means that the animal'simmune system should not be tolerised to that antigen and therefore canmount an immune response against ICOS when injected as recombinantprotein or using cell lines or vesicles expressing ICOS. The immunerepertoire of the knock out animal should contain lymphocytes able torecognise the ICOS protein from that animal species. A non-human testanimal (e.g., a mouse) immunised with human ICOS may thus generateantibodies that bind both human ICOS and the test animal ICOS (e.g.,mouse ICOS).

This has at least two advantages. First, a species cross-reactiveantibody can be used for pre-clinical testing in the non-human testanimal before being taken forward into development in human clinicaltrials. Second, a knock out animal's immune system may be able torecognise a greater number of possible epitopes on a human ICOS moleculecompared with those recognised by an ICOS-expressing animal, so that theimmune repertoire of the knock out animal may contain a greaterfunctional diversity of antibodies. Since there is similarity betweenthe sequences of homologous ICOS molecules from different species, theimmune system of a non-human animal may ordinarily be tolerised to thoseregions of the human ICOS protein that match those of the non-humananimal ICOS, whereas this tolerisation does not occur in a knock outanimal.

The ability to use an ICOS knock out animal, and its advantage forgenerating cross-reactive antibodies, is shown in the Examples. It isparticularly surprising that an ICOS knock out animal could besuccessfully immunised to produce an antibody response, because ICOSitself is involved in the immune system biology such as formation andmaintenance of the germinal centers and contributes to the generation ofan immune response through its role on T follicular helper cells whichare ICOS+ve cells [37]. With this in mind, an ICOS knock out animalmight be predicted to generate a poor antibody response at best.Surprisingly, strong titres were obtained in ICOS knock out mice, andhighly functional antibodies were isolated from among the antibodyrepertoire, including desirable cross-reactive antibodies.

Exemplary embodiments of the invention are set out in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain aspects and embodiments of the invention will now be describedin more detail with reference to the accompanying drawings.

FIG. 1A and FIG. 1B: Determination of serum titres of ICOS KO and wildtype Kymouse against both human and mouse ICOS expressed on CHO cells byflow cytometry. Data illustrate ability of immunoglobulin in sera of (a)ICOS KO mice (KO) or (b) wild type non-ICOS KO mice (HK or HL), eachimmunised with human ICOS expressing MEF cells and human ICOS protein,to bind human ICOS (human ICOS binding) or mouse ICOS (mouse ICOSbinding) expressed on CHO cells. Geometric mean is a measure offluorescent intensity of immunoglobulin binding to cells as determinedby flow cytometry.

FIG. 2: Human ICOS-ligand neutralisation HTRF with human ICOS receptor.Neutralisation profiles of STIM001 to STIM009 anti-ICOS mAbs in humanIgG1 format compared to C398.4A and respective isotype controls. Datarepresentative of four experiments.

FIG. 3: Mouse ICOS-Ligand neutralisation HTRF with mouse ICOS receptor.Neutralisation profiles of STIM001 to STIM009 anti-ICOS mAbs in humanIgG1 format compared to C398.4A and respective isotype controls. Datarepresentative of three experiments.

FIG. 4: Human ICOS-Ligand direct neutralisation HTRF with human ICOSreceptor. Neutralisation profiles of STIM001 to STIM009 anti-ICOS mAbsin human IgG4.PE format compared to C398.4A and respective isotypecontrols. Data representative of four experiments.

FIG. 5: Mouse ICOS-Ligand neutralisation HTRF with mouse ICOS receptor.Neutralisation profiles of STIM001 to STIM009 anti-ICOS mAbs in humanIgG4.PE format compared to C398.4A and respective isotype controls. Datarepresentative of four experiments.

FIG. 6A: Concentration-dependent study of STIM001-mediated ADCC on MJcells by using freshly isolated NK cells as effector cells. The effectorcells and target cells (effector:target ratio of 5:1) were incubatedtogether with antibody for 2 hours. BATDA releasing from lysed targetcells was measured as described in the manufacturer kit instruction. HCis the hybrid isotype control.

FIG. 6B, FIG. 6C, and FIG. 6D: Concentration-dependent study of STIM001and STIM003-mediated ADCC on MJ cells with freshly isolated NK cells aseffector cells. The effector cells and target cells (effector:targetratio of 5:1) were incubated together with antibody for 2 hours. BATDAreleasing from lysed target cells was measured as described in themanufacturer kit instruction. HC is the hybrid isotype control.

FIG. 6E, FIG. 6F, and FIG. 6G: Concentration-dependent study of STIM001(hIgG1) and STIM003 (hIgG1)-mediated ADCC on ICOS-transfected CCRF-CEMcells with freshly isolated NK cells as effector cells. The effectorcells and target cells (effector:target ratio of 5:1) were incubatedtogether with antibody for 4 hours. BATDA releasing from lysed targetcells was measured as described in the manufacturer kit instruction. HCis the hybrid isotype control.

FIG. 7, FIG. 8, and FIG. 9: Anti-ICOS antibody inhibits CT26 tumourgrowth and improved survival when dosed as monotherapy or in combinationwith anti-PDL1. The STIM001 mIgG2a is more potent than the mIgG1 format.The number of animals cured or with stable disease is indicated on eachgraph.

FIG. 10: 2×2 combinations CT26 in vivo efficacy study. Each treatmentgroups is represented by a “spider plot” showing the tumour size ofindividual animals (n=10 per groups). When combined with anti-PDL1antibodies, STIM001 delays tumour growth and improves the survival oftreated animals. The efficacy observed in the presence of STIM001 mIgG2ais superior to that of STIM001 mIgG1. Finally, STIM001 mIgG2a incombination with anti-PDL1 mIgG2a was the most potent combination totrigger the anti-tumour response resulting in 60% of the animals curedof the disease. For each group, the number of animals cured of theirdisease is indicated on the top right of the respective graphs. Dosingwas on days 6, 8, 10, 13, 15 and 17.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, and FIG. 11F: Graphsshowing the CT26 tumour volumes over time of animals treated withanti-ICOS or anti-PDL1 monotherapies or combination therapies. Eachtreatment group is represented by a “spider plot” showing the tumoursize of individual animals (n=10 per group). For each group, the numberof animals with tumour size below 100 mm^3 (stable/cured of theirdisease) is indicated on the top right of the respective graphs. Dosingwas performed on days 6, 8, 10, 13, 15 and 17. Dosing time is indicatedby the shaded area. (FIG. 11A) Isotype control; (FIG. 11B) Anti-PDL1mIgG2a AbW; (FIG. 11C) Anti ICOS STIM003 mIgG1; (FIG. 11D) Anti ICOSSTIM003 mIgG2a; (FIG. 11E) Anti-PDL1 mIgG2a AbW+STIM003 mIgG1; (FIG.11F) Anti-PDL1 mIgG2a AbW+STIM003 mIgG2a. STIM003 mIgG2 significantlyinhibits CT26 tumour growth when combined with anti-PDL1 (AbW) mIgG2a.

FIG. 12: MJ cell in vitro activation assay—bead bound. Stimulationprofiles of STIM001, STIM002 and STIM003 anti-ICOS mAbs bound to beadscompared with anti-ICOS C398.4A and respective isotype controls. Datarepresent the average of two experiments (n=1 in the case of C398.4Aisotype control beads).

FIG. 13: MJ cell in vitro activation assay—plate bound. Stimulationprofiles of plate-bound STIM001, STIM002, STIM003 and STIM004 anti-ICOSmAbs compared with anti-ICOS C398.4A and respective isotype controls.Data represent the average of two experiments.

FIG. 14: FACS analysis of STIM001 and STIM003 hIgG1 binding to activatedT cells. (a) shows a representative experiment of the dose response ofpre-labelled antibodies binding to activated T cells, whereas (b) showsthe binding following the dose response of naked antibodies followed bythe detection with a secondary labelled antibody. Tables indicaterelevant EC50 (M) as determined using GraphPad Prism.

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F, and FIG.15G: STIM001 and STIM003 showed isotype-dependent effects on the T cellcompartment at the tumour site. A total of 1×10E5 CT-26 tumour cellswere implanted subcutaneously in Balb/c female mice. At day 13 and day15 post implantation animals were dosed with antibodies or salineintraperitoneally (n=10/each group). On day 16 post implantation spleenand tumours were harvest from tumour bearing animals (n=8/each group),dissociated and stained for FACS analysis. FIG. 15A, percentage of CD3cells that are positive for CD4 cells. FIG. 15B, percentage of CD3 cellsthat are positive for CD8 cells. FIG. 15C, percentage of CD4 cells thatare Foxp3+ & CD25+. FIG. 15D, percentage of CD4 cells in spleen that arepositive for Foxp3+ & CD25+. FIG. 15E, percentage of CD4 effector cellsin total CD4 cells. FIG. 15F, ratio of CD8 effector to T-Reg cells. FIG.15G, ratio of CD4 effector to T-Reg cells. Statistical analysis wereperformed using GraphPad Prism, all the Antibody treated groups werecompared with saline treated group, P values were noted when significant(p<0.05). Values denote means+SD (n=8 mice/group). For F: Values denotemean+SEM.

FIG. 16: Example data from concentration-dependent study of STIM001(hIgG1) and STIM003 (hIgG1) agonist effect on isolated human T-cellsco-stimulated with anti-CD3/anti-CD28 dynabeads for 3-days in T cellactivation assay 1 (see Example 9b). IFN-γ production was used as anindicator of the agonistic effect. STIM001 (hIgG1) and STIM003 (hIgG1)were tested in plate-bound, soluble or crosslinked soluble (Fc-linkedAb) formats and compared with a hybrid isotype control (HC hIgG1).Included for comparison in the plate-bound assay was hamster antibodyC398.4A and its isotype control (hamster IgG). Upper panel shows datafrom plate-bound antibodies. Lower panel shows data from IgG1 antibodiesin soluble and cross-linked forms. Left and right panels respectivelyuse T cells from two independent human donors.

FIG. 17A and FIG. 17B: Example data set for STIM001 in T cell activationassay 1 (see Example 9). Data indicate levels of IFN-γ induced bySTIM001 (hIgG1) or its hybrid isotype control (HC IgG1) at one givendose for T cells from 8 independent human donors. Plate-bound antibody(FIG. 17A) was used at 5 μg/ml. Soluble antibody (FIG. 17B) was used at15 μg/ml. Each dot represents one donor, identified by number (D214 forexample). Significance was assessed using Wilcoxon statistic test: *,p<0.05 and **, p<0.01.

FIG. 18A and FIG. 18B: Example data set for STIM003 in T cell activationassay 1 (see Example 9). Data indicate levels of IFN-γ induced bySTIM003 (hIgG1) or its hybrid isotype control (HC hIgG1) at one givendose for T cells from 8 independent human healthy donors. Solubleantibody (FIG. 18A) was used at 15 μg/ml. Plate-bound antibody (FIG.18B) was used at 5 μg/ml. Each dot represents one donor, identified bynumber (D214 for example). Significance was assessed using Wilcoxonstatistic test: *, p<0.05 and **, p<0.01.

FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E, and FIG. 19F: Exampledata from T cell activation assay 2 (see Example 9c). Study of STIM001(hIgG1) and STIM003 (hIgG1) agonist effect on isolated human T-cellsstimulated with anti-CD3/anti-CD28 dynabeads for 3-days, then rested inmedium for 3-days and finally re-stimulated with plate-bound STIM001,STIM003 or C398.4A Ab+/−CD3 Ab. Data comparing levels of IFN-γ (FIG. 19Aand FIG. 19B), TNF-α (FIG. 19C and FIG. 19D) and IL-2 (FIG. 19E and FIG.19F) induced by STIM001, STIM003 vs their hybrid control IgG1 (FIG. 19A,FIG. 19C, and FIG. 19E) or C398.4A vs its hamster IgG control (FIG. 19B,FIG. 19D, and FIG. 19F) at one given dose and in combination with CD3 Ab(TCR engagement). Each dot represents an independent donor identifiableby its number (D190 for example). Statistical significance between theAbs and their isotype control was assessed using Wilcoxon statistic testand p value indicated. Note that STIM003 concentration was slightlydifferent to those of HC IgG1 (5.4 vs 5 μg/ml).

FIG. 20: Graph showing the percentage of immune cells (CD8 T-Effector,CD4 T-Effector and CD4/FoxP3 TReg cells) in the CT26 tumours and in thespleen of tumour bearing animals that are expressing ICOS on theirsurface. Values denote mean±SD (n=8). P values were calculated usingnonparametric Dunn's multiple comparisons test. NS=not significant;***=p<0.001; ****=p<0.0001.

FIG. 21: Relative expression of ICOS on the surface of immune cells—CD8T effector, CD4 T effector and CD4/FoxP3 TRegs—as determined by the meanfluorescence intensity (MFI). Values denote mean±SD (n=8). P values werecalculated using nonparametric Dunn's multiple comparisons test.****=p<0.0001, **=p<0.01. Note the difference in the fluorescenceintensity between spleen (low) and tumours (high).

FIG. 22: Effect of STIM001 and STIM003 on the percentage of differentimmune cells in the microenvironment of CT26 tumours. *=p<0.05.

FIG. 23: Effect of antibodies STIM001 and STIM003 on the percentage ofregulatory T cells (CD4+/FoxP3+ cells) in the microenvironment of CT26tumours. **=p<0.05, ****=p<0.0001. Values denote mean±SD (n=8). P valueswere calculated using nonparametric Dunn's multiple comparisons test.

FIG. 24: STIM001 and STIM003 mIgG2 significantly increase the CD8effector T cell to TReg ratio and the CD4 effector T cells to TReg ratioin CT26 tumours. The ratio was determined by dividing the percentage ofeffector cells in the tumour by the percentage of regulatory T cells inthe tumour.

FIG. 25: Effect of antibodies on percentage of immune cells in thespleen of CT26 tumour bearing animals.

FIG. 26: Effect of antibodies on percentage of regulatory T cells(CD4+/FoxP3+ cells) in the spleen of CT26 tumour bearing animals.

FIG. 27A CD8 effector:Treg ratio and FIG. 27B CD4:TReg ratio in spleenof CT26 tumour bearing animals.

FIG. 28A and FIG. 28B: Surface staining of AF647-conjugated STIM001,STIM003 and hIgG1 hybrid control (HC IgG1) on activated Mauritiancynomolgus pan T cells. Data from assays using different donor sourcesof T cells are shown in FIG. 28A and FIG. 28B respectively. EC50 valuesare indicated in the table.

FIG. 29: Kaplan Meier curves for CT26 Balb/C model. Shading shows dosingwindow. LogRank p<0.0001.

FIG. 30, FIG. 31, FIG. 32, and FIG. 33: Graphs showing volumes of A20tumours over time in mice for the study described in Example 20. Eachtreatment group is represented by a spider plot showing tumour size inindividual animals, n=8 per group. For each group, the number of animalswith no sign of tumour (indicating cured of disease) is indicated on thebottom left of the graph. Dosing was performed on days 8, 11, 15, 18,22, 25 and 29 post tumour cell implantation and the dosing time isindicated by the grey shaded area. Compared with the control group (FIG.30) and the anti-PD-L1 treatment group (FIG. 31), the STIM001 mIgG2a(FIG. 32) and STIM003 mIgG2a (FIG. 33) treatment groups showedsignificant inhibition of A20 tumour growth.

FIG. 34A, FIG. 34B, and FIG. 34C: Data from CT26 in vivo efficacy studydescribed in Example 11c using combination of anti-PD-L1 mIgG2a antibodywith single vs multiple doses of STIM003 mIgG2a. Each treatment group isrepresented by a “spider plot” showing the tumour size of individualanimals (n=8 per group). For each group, the number of animals cured oftheir disease is indicated on the bottom right of the respective graph.Dosing days for each antibody are indicated by arrows below therespective graphs.

FIG. 35: STIM002 VH (top) and VL (bottom) domain amino acid sequences,showing residues that differ in the corresponding sequences of STIM001,STIM002B and related antibodies CL-61091, CL-64536, CL-64837, CL-64841and CL-64912 and/or in the human germline. Sequence numbering isaccording to IMGT.

FIG. 36: STIM003 VH (top) and VL (bottom) domain amino acid sequences,showing residues that differ in the corresponding sequences of relatedantibodies CL-71642 and CL-74570 and/or in the human germline. Sequencenumbering is according to IMGT. The VL domain of antibody CL-71642obtained from sequencing is shown here without the N terminal residue.From the alignment it can be seen that the full VH domain sequence wouldcomprise an N terminal glutamic acid.

FIG. 37: STIM007 VH (top) and VL (bottom) domain amino acid sequences,showing residues that differ in the corresponding sequences of STIM008and/or in the human germline. Sequence numbering is according to IMGT.

FIG. 38: Effect of STIM003 (anti-ICOS) and AbW (anti-PD-L1) mIgG2aantibodies in the J558 syngeneic model. Each treatment group isrepresented by a “spider plot” showing the tumour size of individualanimals (n=10 or n=8 per group). STIM003 monotherapy demonstrated someefficacy with 3 of 8 animals cured from their disease. Similarlyanti-PDL1 was effective in this model with 6 out of 8 animals cured fromtheir disease by day 37. When combined with anti-PDL1 antibodies,STIM003 mIgG2 fully inhibited tumour growth and improved the survival oftreated animals. For each group, the number of animals cured of theirdisease is indicated on the bottom right of the respective graph. Dosingdays are indicated by dotted lines (day 11, 15, 18, 22, 25 and 29).

FIG. 39A and FIG. 39B: Quantification of ICOS expression (percentage ofpositive cells and relative expression/dMFI) on the different TILS cellsubtypes in the tumour tissue. (FIG. 39A): The % of immune cell subtypesthat are positive for ICOS expression and (FIG. 39B): the ICOS dMFI(relative ICOS expression on ICOS positive cell) of immune cell subtypesof animals treated with saline or anti-PD-L1 or anti-PD-1 surrogateantibodies. The mice were implanted with 100 μl of 1×10□ viable cells/mlon day 0 (n=7 or n=8). The animals were dosed i.p with 130 ug ofantibody on day 13 and day 15. The tissue samples were isolated andanalysed on day 16. CD4+/FOXP3+ cells were only included for the TRegpopulation (right end side graphs) and were excluded from the “effector”CD4 cells (left end side graphs) which are all Foxp3 negative. SeeExample 22.

FIG. 40A, FIG. 40B, and FIG. 40C: Data from A20 in vivo efficacy study.Each treatment group is represented by a “spider plot” showing thetumour size of individual animals (n=10 per group). For each group, thenumber of animals cured of their disease is indicated on the respectivegraph. For the multiple dose, dosing was on days 8, 11, 15, 18, 22 and25, indicated by dotted lines. For the single dose, animals receivedinjection IP only on day 8. (FIG. 40A): Saline; (FIG. 40B): STIM003mIgG2a multiple dose; (FIG. 40C): STIM003 mIgG2a single dose. SeeExample 23.

FIG. 41: Kaplan-Meier curves for study reported in Example 23 withSTIM003 mIgG2a 60 μg fixed dose. SD=single dose, day 8. MD=multipledoses BIW from day 8.

FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D, FIG. 42E, FIG. 42F, FIG. 42G,and FIG. 42H: ICOS expression on major T cells subsets (T-reg[CD4+/FoxP3+], CD4 Eff [CD4+/FoxP3-]cells and CD8+) from CT26 tumourbearing animals (n=4 per time point) dosed with saline. Immune cellsphenotyping were conducted on day 1, 2, 3, 4 and 8 post treatment andstained for ICOS expression in all the tissues at all time points. FIG.42A, FIG. 42B, FIG. 42C, and FIG. 42D show the percentage of ICOSpositive cells at all the time points in four different tissues. FIG.42E, FIG. 42F, FIG. 42G, and FIG. 42H show the ICOS dMFI (relativeexpression) all the time points in all the four different tissues. SeeExample 24.

FIG. 43A and FIG. 43B: FACS analysis demonstrating T-reg depletion inthe TME in response to STIM003 mIgG2a antibody. CT-26 tumour bearinganimals were treated with a single dose (6, 60 or 200 μg) of STIM003 onday 12 post tumour cell implantation. Tissues (n=4 per time point) whereharvested for FACS analysis on day 1, 2, 3, 4 and 8 post treatment. Thepercentage of T-reg cells (CD4⁺CD25⁺Foxp3⁺) in total tumour (FIG. 43A)and the percentage of T-reg cells in the blood (FIG. 43B) are shown atthe different time points. See Example 24.

FIG. 44A, FIG. 44B, FIG. 44C, and FIG. 44D: Increase in CD8:T-reg andCD4 eff:T-reg ratio in response to STIM003 mIgG2a. CT-26 tumour bearinganimals received a single dose (6, 60 or 200 μg) of STIM003 mIgG2a onday 12 post tumour cell implantation. Tissues (n=4 per time point) wereharvested for FACS analysis on day 1, 2, 3, 4 and 8 post treatment and Teff to T-reg ratios were calculated. (FIG. 44A) & (FIG. 44B), CD8:T-regratio in tumour and blood, (FIG. 44C) & (FIG. 44D) CD4-eff:T-reg ratioin tumour and blood. See Example 24.

FIG. 45A, FIG. 45B, FIG. 45C, FIG. 45D, FIG. 45E, FIG. 45F, FIG. 45G,and FIG. 45H: STIM003 treatment correlates with increased degranulationand Th1 cytokine production by TILs. On day 8 post treatment TILs wereisolated and FACS analysis were performed to detect CD107a expression onCD4 and CD8 T cells (FIG. 45A and FIG. 45B). In parallel, cells fromdissociated tumours were rested for 4 hrs in the presence ofBrefeldin-A, cells were stained for T cells markers and permeabilisedfor intracellular staining to detect IFN-γ and TNF-α (FIG. 45C, FIG.45D, FIG. 45E, FIG. 45F, FIG. 45G, and FIG. 45H). See Example 24.

DETAILED DESCRIPTION

ICOS

Antibodies according to the present invention bind the extracellulardomain of human ICOS. Thus, the antibodies bind ICOS-expressing Tlymphocytes. “ICOS” or “the ICOS receptor” referred to herein may behuman ICOS, unless the context dictates otherwise. Sequences of human,cynomolgus and mouse ICOS are shown in the appended sequence listing,and are available from NCBI as human NCBI ID: NP_036224.1, mouse NCBIID: NP_059508.2 and cynomolgus GenBank ID: EHH55098.1.

Cross-Reactivity

Antibodies according to the present invention are preferablycross-reactive, and may for example bind the extracellular domain ofmouse ICOS as well as human ICOS. The antibodies may bind othernon-human ICOS, including ICOS of primates such as cynomolgus. Ananti-ICOS antibody intended for therapeutic use in humans must bindhuman ICOS, whereas binding to ICOS of other species would not havedirect therapeutic relevance in the human clinical context.Nevertheless, the data herein indicate that antibodies that bind bothhuman and mouse ICOS have properties that render them particularlysuitable as agonist and depleting molecules. This may result from one ormore particular epitopes being targeted by the cross-reactiveantibodies. Regardless of the underlying theory, however, cross-reactiveantibodies are of high value and are excellent candidates as therapeuticmolecules for pre-clinical and clinical studies.

As explained in the experimental Examples, the STIM antibodies describedhere were generated using Kymouse™ technology where the mouse had beenengineered to lack expression of mouse ICOS (an ICOS knock-out). ICOSknock-out transgenic animals and their use for generating cross-reactiveantibodies are further aspects of the present invention.

One way to quantify the extent of species cross-reactivity of anantibody is as the fold-difference in its affinity for antigen or onespecies compared with antigen of another species, e.g., fold differencein affinity for human ICOS vs mouse ICOS. Affinity may be quantified asK_(D), referring to the equilibrium dissociation constant of theantibody-antigen reaction as determined by SPR with the antibody in Fabformat as described elsewhere herein. A species cross-reactive anti-ICOSantibody may have a fold-difference in affinity for binding human andmouse ICOS that is 30-fold or less, 25-fold or less, 20-fold or less,15-fold or less, 10-fold or less or 5-fold or less. To put it anotherway, the K_(D) of binding the extracellular domain of human ICOS may bewithin 30-fold, 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of theK_(D) of binding the extracellular domain of mouse ICOS. Antibodies canalso be considered cross-reactive if the K_(D) for binding antigen ofboth species meets a threshold value, e.g., if the K_(D) of bindinghuman ICOS and the K_(D) of binding mouse ICOS are both 10 mM or less,preferably 5 mM or less, more preferably 1 mM or less. The K_(D) may be10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. The K_(D)may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less,0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1nM or less.

An alternative measure of cross-reactivity for binding human ICOS andmouse ICOS is the ability of an antibody to neutralise ICOS ligandbinding to ICOS receptor, such as in an HTRF assay (see Example 8).Examples of species cross-reactive antibodies are provided herein,including STIM001, STIM002, STIM002-B, STIM003, STIM005 and STIM006,each of which was confirmed as neutralising binding of human B7-H2 (ICOSligand) to human ICOS and neutralising binding of mouse B7-H2 to mouseICOS in an HTRF assay. Any of these antibodies or their variants may beselected when an antibody cross-reactive for human and mouse ICOS isdesired. A species cross-reactive anti-ICOS antibody may have an IC50for inhibiting binding of human ICOS to human ICOS receptor that iswithin 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of the IC50 forinhibiting mouse ICOS to mouse ICOS receptor as determined in an HTRFassay. Antibodies can also be considered cross-reactive if the IC50 forinhibiting binding of human ICOS to human ICOS receptor and the IC50 forinhibiting binding of mouse ICOS to mouse ICOS receptor are both 1 mM orless, preferably 0.5 mM or less, e.g., 30 nM or less, 20 nM or less, 10nM or less. The IC50s may be 5 nM or less, 4 nM or less, 3 nM or less or2 nM or less. In some cases the IC50s will be at least 0.1 nM, at least0.5 nM or at least 1 nM.

Specificity

Antibodies according to the present invention are preferably specificfor ICOS. That is, the antibody binds its epitope on the target protein,ICOS (human ICOS, and preferably mouse and/or cynomolgus ICOS as notedabove), but does not show significant binding to molecules that do notpresent that epitope, including other molecules in the CD28 gene family.An antibody according to the present invention preferably does not bindhuman CD28. The antibody preferably also does not bind mouse orcynomolgus CD28.

CD28 co-stimulates T cell responses when engaged by its ligands CD80 andCD86 on professional antigen presenting cells in the context of antigenrecognition via the TCR. For various in vivo uses of the antibodiesdescribed herein, the avoidance of binding to CD28 is consideredadvantageous. Non-binding of the anti-ICOS antibody to CD28 should allowCD28 to interact with its native ligands and to generate appropriateco-stimulatory signal for T cell activation. Additionally, non-bindingof the anti-ICOS antibody to CD28 avoids the risk of superagonism.Over-stimulation of CD28 can induce proliferation in resting T cellswithout the normal requirement for recognition of a cognate antigen viathe TCR, potentially leading to runaway activation of T cells andconsequent cytokine-release syndrome, especially in human subjects. Thenon-recognition of CD28 by antibodies according to the present inventiontherefore represents an advantage in terms of their safe clinical use inhumans.

As discussed elsewhere herein, the present invention extends tomultispecific antibodies (e.g., bispecifics). A multispecific (e.g.,bispecific) antibody may comprise (i) an antibody antigen binding sitefor ICOS and (ii) a further antigen binding site (optionally an antibodyantigen binding site, as described herein) which recognises anotherantigen (e.g., PD-L1). Specific binding of individual antigen bindingsites may be determined. Thus, antibodies that specifically bind ICOSinclude antibodies comprising an antigen binding site that specificallybinds ICOS, wherein optionally the antigen binding site for ICOS iscomprised within an antigen-binding molecule that further includes oneor more additional binding sites for one or more other antigens, e.g., abispecific antibody that binds ICOS and PD-L1.

Affinity

The affinity of binding of an antibody to ICOS may be determined.Affinity of an antibody for its antigen may be quantified in terms ofthe equilibrium dissociation constant K_(D), the ratio Ka/Kd of theassociation or on-rate (Ka) and the dissociation or off-rate (kd) of theantibody-antigen interaction. Kd, Ka and Kd for antibody-antigen bindingcan be measured using surface plasmon resonance (SPR).

An antibody according to the present invention may bind the EC domain ofhuman ICOS with a K_(D) of 10 mM or less, preferably 5 mM or less, morepreferably 1 mM or less. The K_(D) may be 50 nM or less, 10 nM or less,5 nM or less, 2 nM or less, or 1 nM or less. The K_(D) may be 0.9 nM orless, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less,0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. TheK_(D) may be at least 0.001 nM, for example at least 0.01 nM or at least0.1 nM.

Quantification of affinity may be performed using SPR with the antibodyin Fab format. A suitable protocol is as follows:

1. Coupling anti-human (or other antibody constant regionspecies-matched) IgG to a biosensor chip (e.g., GLM chip) such as byprimary amine coupling;

2. Exposing the anti-human IgG (or other matched species antibody) to atest antibody, e.g., in Fab format, to capture test antibody on thechip;

3. Passing the test antigen over the chip's capture surface at a rangeof concentrations, e.g., at 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM and 2nM, and at 0 nM (i.e., buffer alone); and

4. Determining the affinity of binding of test antibody to test antigenusing SPR at 25° C. Buffer may be at pH 7.6, 150 mM NaCl, 0.05%detergent (e.g., P20) and 3 mM EDTA. Buffer may optionally contain 10 mMHEPES. HBS-EP can be used as running buffer. HBS-EP is available fromTeknova Inc (California; catalogue number H8022).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH 1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, e.g., using amodel inherent to the ProteOn XPR36™ analysis software.

A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™(Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc). Worked examples ofSPR are found in Example 7.

As described, affinity may be determined by SPR with the antibody in Fabformat, with the antigen coupled to the chip surface and the testantibody passed over the chip in Fab format in solution, to determineaffinity of the monomeric antibody-antigen interaction. Affinity can bedetermined at any desired pH, e.g., pH 5.5 or pH 7.6, and any desiredtemperature e.g., 25° C. or 37° C. As reported in Example 7, antibodiesaccording to the present invention bound human ICOS with an apparentaffinity of less than 2 nM, as determined by SPR using the antibody inmonovalent (Fab) format.

Other ways to measure binding of an antibody to ICOS includefluorescence activated cell sorting (FACS), e.g., using cells (e.g., CHOcells) with exogenous surface expression of ICOS or activated primary Tcells expressing endogenous levels of ICOS. Antibody binding toICOS-expressing cells as measured by FACS indicates that the antibody isable to bind the extracellular (EC) domain of ICOS.

ICOS Receptor Agonism

The ICOS ligand (ICOSL, also known as B7-H2) is a cell surface expressedmolecule that binds to the ICOS receptor [17]. This intercellularligand-receptor interaction promotes multimerisation of ICOS on the Tcell surface, activating the receptor and stimulating downstreamsignalling in the T cell. In effector T cells, this receptor activationstimulates the effector T cell response.

Anti-ICOS antibodies may act as agonists of ICOS, mimicking and evensurpassing this stimulatory effect of the native ICOS ligand on thereceptor. Such agonism may result from ability of the antibody topromote multimerisation of ICOS on the T cell. One mechanism for this iswhere the antibodies form intercellular bridges between ICOS on the Tcell surface and receptors on an adjacent cell (e.g., B cell,antigen-presenting cell, or other immune cell), such as Fc receptors.Another mechanism is where antibodies having multiple (e.g., two)antigen-binding sites (e.g., two VH-VL domain pairs) bridge multipleICOS receptor molecules and so promote multimerisation. A combination ofthese mechanisms may occur.

Agonism can be tested for in in vitro T cell activation assays, usingantibody in soluble form (e.g., in immunoglobulin format or otherantibody format comprising two spatially separated antigen-bindingsites, e.g., two VH-VL pairs), either including or excluding across-linking agent, or using antibody bound to a solid surface toprovide a tethered array of antigen-binding sites. Agonism assays mayuse a human ICOS positive T lymphocyte cell line such as MJ cells (ATCCCRL-8294) as the target T cell for activation in such assays. One ormore measures of T cell activation can be determined for a test antibodyand compared with a reference molecule or a negative control todetermine whether there is a statistically significant (p<0.05)difference in T cell activation effected by the test antibody comparedwith the reference molecule or the control. One suitable measure of Tcell activation is production of cytokines, e.g., IFNγ, TNFα or IL-2.The skilled person will include suitable controls as appropriate,standardising assay conditions between test antibody and control. Asuitable negative control is an antibody in the same format (e.g.,isotype control) that does not bind ICOS, e.g., an antibody specific foran antigen that is not present in the assay system. A significantdifference is observed for test antibody relative to a cognate isotypecontrol within the dynamic range of the assay is indicative that theantibody acts as an agonist of the ICOS receptor in that assay.

An agonist antibody may be defined as one which, when tested in a T cellactivation assay:

has a significantly lower EC50 for induction of IFNγ production comparedwith control antibody;

induces significantly higher maximal IFNγ production compared withcontrol antibody;

has a significantly lower EC50 for induction of IFNγ production comparedwith ICOSL-Fc;

induces significantly higher maximal IFNγ production compared withICOSL-Fc;

has a significantly lower EC50 for induction of IFNγ production comparedwith reference antibody C398.4A; and/or

induces significantly higher maximal IFNγ production compared withreference antibody C398.4A.

In vitro T cell assays include the bead-bound assay of Example 13, theplate-bound assay of Example 14 and the soluble form assay of Example15.

A significantly lower or significantly higher value may for example beup to 0.5-fold different, up to 0.75-fold different, up to 2-folddifferent, up to 3-fold different, up to 4-fold different or up to5-fold different, compared with the reference or control value.

Thus, in one example, an antibody according to the present invention hasa significantly lower, e.g., at least 2-fold lower, EC50 for inductionof IFNγ in an MJ cell activation assay using the antibody in bead-boundformat, compared with control.

The bead-bound assay uses the antibody (and, for control or referenceexperiments, the control antibody, reference antibody or ICOSL-Fc) boundto the surface of beads. Magnetic beads may be used, and various kindsare commercially available, e.g., Tosyl-activated DYNABEADS M-450 (DYNALInc, 5 Delaware Drive, Lake Success, N.Y. 11042 Prod No. 140.03,140.04). Beads may be coated as described in Example 13, or generally bydissolving the coating material in carbonate buffer (pH 9.6, 0.2 M) orother method known in the art. Use of beads conveniently allows thequantity of protein bound to the bead surface to be determined with agood degree of accuracy. Standard Fc-protein quantification methods canbe used for coupled protein quantification on beads. Any suitable methodcan be used, with reference to a relevant standard within the dynamicrange of the assay. DELFIA is exemplified in Example 13, but ELISA orother methods could be used.

Agonism activity of an antibody can also be measured in primary human Tlymphocytes ex vivo. The ability of an antibody to induce expression ofIFNγ in such T cells is indicative of ICOS agonism. Described herein aretwo T cell activation assays using primary cells—see Example 2, T cellactivation assay 1 and T cell activation assay 2. Preferably, anantibody will show significant (p<0.05) induction of IFNγ at 5 μg/mlcompared with control antibody in T cell activation assay 1 and/or Tcell activation assay 2. As noted above, an anti-ICOS antibody maystimulate T cell activation to a greater degree than ICOS-L or C398.4 insuch an assay. Thus, the antibody may show significantly (p<0.05)greater induction of IFNγ at 5 μg/ml compared with the control orreference antibody in T cell activation assay 1 or 2. TNFα or IL-2induction may be measured as an alternative assay readout.

Agonism of an anti-ICOS antibody may contribute to its ability to changethe balance between populations of TReg and TEff cells in vivo, e.g., ina site of pathology such as a tumour microenvironment, in favour of TEffcells. The ability of an antibody to enhance tumour cell killing byactivated ICOS-positive effector T cells may be determined, as discussedelsewhere herein.

T Cell Dependent Killing

Effector T cell function can be determined in a biologically relevantcontext using an in vitro co-culture assay where tumour cells areincubated with relevant immune cells to trigger immune cell-dependentkilling, in which the effect of an anti-ICOS antibody on tumour cellkilling by TEffs is observed.

The ability of an antibody to enhance tumour cell killing by activatedICOS-positive effector T cells may be determined. An anti-ICOS antibodymay stimulate significantly greater (p<0.05) tumour cell killingcompared with a control antibody. An anti-ICOS antibody may stimulatesimilar or greater tumour cell killing in such an assay as compared witha reference molecule such as the ICOS ligand or the C398.4 antibody. Asimilar degree of tumour cell killing can be represented as the assayreadout for the test antibody being less than two-fold different fromthat for the reference molecule.

ICOS Ligand-Receptor Neutralisation Potency

An antibody according to the present invention may be one which inhibitsbinding of ICOS to its ligand ICOSL.

The degree to which an antibody inhibits binding of the ICOS receptor toits ligand is referred to as its ligand-receptor neutralising potency.Potency is normally expressed as an IC50 value, in pM unless otherwisestated. In ligand-binding studies, IC50 is the concentration thatreduces receptor binding by 50% of maximal specific binding level. IC50may be calculated by plotting % specific receptor binding as a functionof the log of the antibody concentration, and using a software programsuch as Prism (GraphPad) to fit a sigmoidal function to the data togenerate IC50 values. Neutralising potency may be determined in an HTRFassay. A detailed working example of an HTRF assay for ligand-receptorneutralising potency is set out in Example 8.

An IC50 value may represent the mean of a plurality of measurements.Thus, for example, IC50 values may be obtained from the results oftriplicate experiments, and a mean IC50 value can then be calculated.

An antibody may have an IC50 of 1 mM or less in a ligand-receptorneutralisation assay, e.g., 0.5 mM or less. The IC50 may be, 30 nM orless, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM orless or 2 nM or less. The IC50 may be at least 0.1 nM, at least 0.5 nMor at least 1 nM.

Antibodies

As described in more detail in the Examples, we isolated andcharacterised antibodies of particular interest, designated STIM001,STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008and STIM009. In various aspects of the invention, unless contextdictates otherwise, antibodies may be selected from any of theseantibodies, or from the sub-set of STIM001, STIM002, STIM003, STIM004and STIM005. Sequences of each of these antibodies are provided in theappended sequence listing, wherein for each antibody the followingsequences are shown: nucleotide sequence encoding VH domain; amino acidsequence of VH domain; VH CDR1 amino acid sequence, VH CDR2 amino acidsequence; VH CDR3 amino acid sequence; nucleotide sequence encoding VLdomain; amino acid sequence of VL domain; VL CDR1 amino acid sequence;VL CDR2 amino acid sequence; and VL CDR3 amino acid sequence,respectively. The present invention encompasses anti-ICOS antibodieshaving the VH and/or VL domain sequences of all antibodies shown in theappended sequence listing and/or in the drawings, as well as antibodiescomprising the HCDRs and/or LCDRs of those antibodies, and optionallyhaving the full heavy chain and/or full light chain amino acid sequence.

STIM001 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:366, comprising the CDRH1 amino acid sequence of Seq IDNo:363, the CDRH2 amino acid sequence of Seq ID No:364, and the CDRH3amino acid sequence of Seq ID No:365. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:367. STIM001 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:373, comprisingthe CDRL1 amino acid sequence of Seq ID No:370, the CDRL2 amino acidsequence of Seq ID No:371, and the CDRL3 amino acid sequence of Seq IDNo:372. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:374. The V_(H) domain may be combined with any of the heavy chainconstant region sequences described herein, e.g. Seq ID No:193, Seq IDNo:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, SeqID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528,Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavychain amino acid sequence is Seq ID No:368 (heavy chain nucleic acidsequence Seq ID No:369). A full length light chain amino acid sequenceis Seq ID No:375 (light chain nucleic acid sequence Seq ID No:376).

STIM002 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:380, comprising the CDRH1 amino acid sequence of Seq IDNo:377, the CDRH2 amino acid sequence of Seq ID No:378, and the CDRH3amino acid sequence of Seq ID No:379. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:381. STIM002 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:387, comprisingthe CDRL1 amino acid sequence of Seq ID No:384, the CDRL2 amino acidsequence of Seq ID No:385, and the CDRL3 amino acid sequence of Seq IDNo:386. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:388 or Seq ID No:519. The V_(H) domain may be combined with any ofthe heavy chain constant region sequences described herein, e.g. Seq IDNo:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, SeqID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526,Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L)domain may be combined with any of the light chain constant regionsequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. Afull length heavy chain amino acid sequence is Seq ID No:382 (heavychain nucleic acid sequence Seq ID No:383). A full length light chainamino acid sequence is Seq ID No:389 (light chain nucleic acid sequenceSeq ID No:390 or Seq ID NO:520).

STIM002-B has a heavy chain variable region (V_(H)) amino acid sequenceof Seq ID No:394, comprising the CDRH1 amino acid sequence of Seq IDNo:391, the CDRH2 amino acid sequence of Seq ID No:392, and the CDRH3amino acid sequence of Seq ID No:393. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:395. STIM002-B has a lightchain variable region (V_(L)) amino acid sequence of Seq ID No:401,comprising the CDRL1 amino acid sequence of Seq ID No:398, the CDRL2amino acid sequence of Seq ID No:399, and the CDRL3 amino acid sequenceof Seq ID No:400. The light chain nucleic acid sequence of the V_(L)domain is Seq ID No:402. The V_(H) domain may be combined with any ofthe heavy chain constant region sequences described herein, e.g. Seq IDNo:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, SeqID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526,Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L)domain may be combined with any of the light chain constant regionsequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. Afull length heavy chain amino acid sequence is Seq ID No:396 (heavychain nucleic acid sequence Seq ID No:397). A full length light chainamino acid sequence is Seq ID No:403 (light chain nucleic acid sequenceSeq ID No:404).

STIM003 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:408, comprising the CDRH1 amino acid sequence of Seq IDNo:405, the CDRH2 amino acid sequence of Seq ID No:406, and the CDRH3amino acid sequence of Seq ID No:407. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:409 or Seq ID No:521. STIM003has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:415, comprising the CDRL1 amino acid sequence of Seq ID No:412, theCDRL2 amino acid sequence of Seq ID No:413, and the CDRL3 amino acidsequence of Seq ID No:414. The light chain nucleic acid sequence of theV_(L) domain is Seq ID No:4416. The V_(H) domain may be combined withany of the heavy chain constant region sequences described herein, e.g.Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq IDNo:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, SeqID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534.The V_(L) domain may be combined with any of the light chain constantregion sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538.A full length heavy chain amino acid sequence is Seq ID No:410 (heavychain nucleic acid sequence Seq ID No:411 or Seq ID No:522). A fulllength light chain amino acid sequence is Seq ID No:417 (light chainnucleic acid sequence Seq ID No:418).

STIM004 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:422, comprising the CDRH1 amino acid sequence of Seq IDNo:419, the CDRH2 amino acid sequence of Seq ID No:420, and the CDRH3amino acid sequence of Seq ID No:421. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:423. STIM004 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:429, comprisingthe CDRL1 amino acid sequence of Seq ID No:426, the CDRL2 amino acidsequence of Seq ID No:427, and the CDRL3 amino acid sequence of Seq IDNo:428. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:430 or Seq ID No:431. The V_(H) domain may be combined with any ofthe heavy chain constant region sequences described herein, e.g. Seq IDNo:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, SeqID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526,Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L)domain may be combined with any of the light chain constant regionsequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. Afull length heavy chain amino acid sequence is Seq ID No:424 (heavychain nucleic acid sequence Seq ID No:425). A full length light chainamino acid sequence is Seq ID No:432 (light chain nucleic acid sequenceSeq ID No:433 or Seq ID no: 434).

STIM005 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:438, comprising the CDRH1 amino acid sequence of Seq IDNo:435, the CDRH2 amino acid sequence of Seq ID No:436, and the CDRH3amino acid sequence of Seq ID No:437. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:439. STIM005 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:445, comprisingthe CDRL1 amino acid sequence of Seq ID No:442, the CDRL2 amino acidsequence of Seq ID No:443, and the CDRL3 amino acid sequence of Seq IDNo:444. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:446. The V_(H) domain may be combined with any of the heavy chainconstant region sequences described herein, e.g. Seq ID No:193, Seq IDNo:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, SeqID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528,Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavychain amino acid sequence is Seq ID No:440 (heavy chain nucleic acidsequence Seq ID No:441). A full length light chain amino acid sequenceis Seq ID No:447 (light chain nucleic acid sequence Seq ID No:448).

STIM006 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:452, comprising the CDRH1 amino acid sequence of Seq IDNo:449, the CDRH2 amino acid sequence of Seq ID No:450, and the CDRH3amino acid sequence of Seq ID No:451. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:453. STIM006 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:459, comprisingthe CDRL1 amino acid sequence of Seq ID No:456, the CDRL2 amino acidsequence of Seq ID No:457, and the CDRL3 amino acid sequence of Seq IDNo:458. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:460. The V_(H) domain may be combined with any of the heavy chainconstant region sequences described herein, e.g. Seq ID No:193, Seq IDNo:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, SeqID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528,Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavychain amino acid sequence is Seq ID No:454 (heavy chain nucleic acidsequence Seq ID No:455). A full length light chain amino acid sequenceis Seq ID No:461 (light chain nucleic acid sequence Seq ID No:462).

STIM007 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:466, comprising the CDRH1 amino acid sequence of Seq IDNo:463, the CDRH2 amino acid sequence of Seq ID No:464, and the CDRH3amino acid sequence of Seq ID No:465. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:467. STIM007 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:473, comprisingthe CDRL1 amino acid sequence of Seq ID No:470, the CDRL2 amino acidsequence of Seq ID No:471, and the CDRL3 amino acid sequence of Seq IDNo:472. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:474. The V_(H) domain may be combined with any of the heavy chainconstant region sequences described herein, e.g. Seq ID No:193, Seq IDNo:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, SeqID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528,Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavychain amino acid sequence is Seq ID No:468 (heavy chain nucleic acidsequence Seq ID No:469). A full length light chain amino acid sequenceis Seq ID No:475 (light chain nucleic acid sequence Seq ID No:476).

STIM008 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:480, comprising the CDRH1 amino acid sequence of Seq IDNo:477, the CDRH2 amino acid sequence of Seq ID No:478, and the CDRH3amino acid sequence of Seq ID No:479. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:481. STIM008 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:487, comprisingthe CDRL1 amino acid sequence of Seq ID No:484, the CDRL2 amino acidsequence of Seq ID No:485, and the CDRL3 amino acid sequence of Seq IDNo:486. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:488. The V_(H) domain may be combined with any of the heavy chainconstant region sequences described herein, e.g. Seq ID No:193, Seq IDNo:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, SeqID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528,Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavychain amino acid sequence is Seq ID No:482 (heavy chain nucleic acidsequence Seq ID No:483). A full length light chain amino acid sequenceis Seq ID No:489 (light chain nucleic acid sequence Seq ID No:490).

STIM009 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:494, comprising the CDRH1 amino acid sequence of Seq IDNo:491, the CDRH2 amino acid sequence of Seq ID No:492, and the CDRH3amino acid sequence of Seq ID No:493. The heavy chain nucleic acidsequence of the V_(H) domain is Seq ID No:495. STIM009 has a light chainvariable region (V_(L)) amino acid sequence of Seq ID No:501, comprisingthe CDRL1 amino acid sequence of Seq ID No:498, the CDRL2 amino acidsequence of Seq ID No:499, and the CDRL3 amino acid sequence of Seq IDNo:500. The light chain nucleic acid sequence of the V_(L) domain is SeqID No:502. The V_(H) domain may be combined with any of the heavy chainconstant region sequences described herein, e.g. Seq ID No:193, Seq IDNo:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, SeqID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528,Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavychain amino acid sequence is Seq ID No:496 (heavy chain nucleic acidsequence Seq ID No:497). A full length light chain amino acid sequenceis Seq ID No:503 (light chain nucleic acid sequence Seq ID No:504).

Antibodies according to the present invention are immunoglobulins ormolecules comprising immunoglobulin domains, whether natural or partlyor wholly synthetically produced. Antibodies may be IgG, IgM, IgA, IgDor IgE molecules or antigen-specific antibody fragments thereof(including, but not limited to, a Fab, F(ab′)2, Fv, disulphide linkedFv, scFv, single domain antibody, closed conformation multispecificantibody, disulphide-linked scfv, diabody), whether derived from anyspecies that naturally produces an antibody, or created by recombinantDNA technology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria. Antibodies can be humanised usingroutine technology. The term antibody covers any polypeptide or proteincomprising an antibody antigen-binding site. An antigen-binding site(paratope) is the part of an antibody that binds to and is complementaryto the epitope of its target antigen (ICOS).

The term “epitope” refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The antigen binding site is a polypeptide or domain that comprises oneor more CDRs of an antibody and is capable of binding the antigen. Forexample, the polypeptide comprises a CDR3 (e.g., HCDR3). For example thepolypeptide comprises CDRs 1 and 2 (e.g., HCDR1 and 2) or CDRs 1-3 of avariable domain of an antibody (e.g., HCDRs1-3).

An antibody antigen-binding site may be provided by one or more antibodyvariable domains. In an example, the antibody binding site is providedby a single variable domain, e.g., a heavy chain variable domain (VHdomain) or a light chain variable domain (VL domain). In anotherexample, the binding site comprises a VH/VL pair or two or more of suchpairs. Thus, an antibody antigen-binding site may comprise a VH and aVL.

The antibody may be a whole immunoglobulin, including constant regions,or may be an antibody fragment. An antibody fragment is a portion of anintact antibody, for example comprising the antigen binding and/orvariable region of the intact antibody. Examples of antibody fragmentsinclude:

(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment includingtwo Fab fragments linked by a disulfide bridge at the hinge region;

(iii) an Fd fragment consisting of the VH and CH1 domains;

(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody,

(v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which isincorporated by reference herein in its entirety), which consists of aVH or VL domain; and

(vi) an isolated complementarity determining region (CDR) that retainsspecific antigen-binding functionality.

Further examples of antibodies are H2 antibodies that comprise a dimerof a heavy chain (5′-VH-(optional hinge)-CH2-CH3-3′) and are devoid of alight chain.

Single-chain antibodies (e.g., scFv) are a commonly used fragment.Multispecific antibodies may be formed from antibody fragments. Anantibody of the invention may employ any such format, as appropriate.

Optionally, the antibody immunoglobulin domains may be fused orconjugated to additional polypeptide sequences and/or to labels, tags,toxins or other molecules. Antibody immunoglobulin domains may be fusedor conjugated to one or more different antigen binding regions,providing a molecule that is able to bind a second antigen in additionto ICOS. An antibody of the present invention may be a multispecificantibody, e.g., a bispecific antibody, comprising (i) an antibodyantigen binding site for ICOS and (ii) a further antigen binding site(optionally an antibody antigen binding site, as described herein) whichrecognises another antigen (e.g., PD-L1).

An antibody normally comprises an antibody VH and/or VL domain. IsolatedVH and VL domains of antibodies are also part of the invention. Theantibody variable domains are the portions of the light and heavy chainsof antibodies that include amino acid sequences of complementaritydetermining regions (CDRs; ie., CDR1, CDR2, and CDR3), and frameworkregions (FRs). Thus, within each of the VH and VL domains are CDRs andFRs. A VH domain comprises a set of HCDRs, and a VL domain comprises aset of LCDRs. VH refers to the variable domain of the heavy chain. VLrefers to the variable domain of the light chain. Each VH and VL istypically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. According to the methods used in thisinvention, the amino acid positions assigned to CDRs and FRs may bedefined according to Kabat (Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1987 and 1991))or according to IMGT nomenclature. An antibody may comprise an antibodyVH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It mayalternatively or also comprise an antibody VL domain comprising a VLCDR1, CDR2 and CDR3 and a framework. Examples of antibody VH and VLdomains and CDRs according to the present invention are as listed in theappended sequence listing that forms part of the present disclosure. TheCDRs shown in the sequence listing are defined according to the IMGTsystem [18]. All VH and VL sequences, CDR sequences, sets of CDRs andsets of HCDRs and sets of LCDRs disclosed herein represent aspects andembodiments of the invention. As described herein, a “set of CDRs”comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1,HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3.Unless otherwise stated, a “set of CDRs” includes HCDRs and LCDRs.

An antibody the invention may comprise one or more CDRs as describedherein, e.g. a CDR3, and optionally also a CDR1 and CDR2 to form a setof CDRs. The CDR or set of CDRs may be a CDR or set of CDRs of any ofSTIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006,STIM007, STIM008 and STIM009, or may be a variant thereof as describedherein.

The invention provides antibodies comprising an HCDR1, HCDR2 and/orHCDR3 of any of antibodies STIM001, STIM002, STIM002-B, STIM003,STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 and/or an LCDR1,LCDR2 and/or LCDR3 of any of these antibodies, e.g. a set of CDRs. Theantibody may comprise a set of VH CDRs of one of these antibodies.Optionally it may also comprise a set of VL CDRs of one of theseantibodies, and the VL CDRs may be from the same or a different antibodyas the VH CDRs.

A VH domain comprising a disclosed set of HCDRs, and/or a VL domaincomprising a disclosed set of LCDRs, are also provided by the invention.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below a VH or VLdomain alone may be used to bind antigen. The STIM003 VH domain may bepaired with the STIM003 VL domain, so that an antibody antigen-bindingsite is formed comprising both the STIM003 VH and VL domains. Analogousembodiments are provided for the other VH and VL domains disclosedherein. In other embodiments, the STIM003 VH is paired with a VL domainother than the STIM003 VL. Light-chain promiscuity is well establishedin the art. Again, analogous embodiments are provided by the inventionfor the other VH and VL domains disclosed herein.

Thus, the VH of any of antibodies STIM001, STIM002, STIM003, STIM004 andSTIM005 may be paired with the VL of any of antibodies STIM001, STIM002,STIM003, STIM004 and STIM005. Further, the VH of any of antibodiesSTIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006,STIM007, STIM008 and STIM009 may be paired with the VL of any ofantibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 or STIM009.

An antibody may comprise one or more CDRs, e.g. a set of CDRs, within anantibody framework. The framework regions may be of human germline genesegment sequences. Thus, the antibody may be a human antibody having aVH domain comprising a set of HCDRs in a human germline framework.Normally the antibody also has a VL domain comprising a set of LCDRs,e.g. in a human germline framework. An antibody “gene segment”, e.g., aVH gene segment, D gene segment, or JH gene segment refers tooligonucleotide having a nucleic acid sequence from which that portionof an antibody is derived, e.g., a VH gene segment is an oligonucleotidecomprising a nucleic acid sequence that corresponds to a polypeptide VHdomain from FR1 to part of CDR3. Human V, D and J gene segmentsrecombine to generate the VH domain, and human V and J segmentsrecombine to generate the VL domain. The D domain or region refers tothe diversity domain or region of an antibody chain. J domain or regionrefers to the joining domain or region of an antibody chain. Somatichypermutation may result in an antibody VH or VL domain having frameworkregions that do not exactly match or align with the corresponding genesegments, but sequence alignment can be used to identify the closestgene segments and thus identify from which particular combination ofgene segments a particular VH or VL domain is derived. When aligningantibody sequences with gene segments, the antibody amino acid sequencemay be aligned with the amino acid sequence encoded by the gene segment,or the antibody nucleotide sequence may be aligned directly with thenucleotide sequence of the gene segment.

Alignments of STIM antibody VH and VL domain sequences against relatedantibodies and against human germline sequences are shown in FIG. 35,FIG. 36 and FIG. 37.

An antibody of the invention may be a human antibody or a chimaericantibody comprising human variable regions and non-human (e.g., mouse)constant regions. The antibody of the invention for example has humanvariable regions, and optionally also has human constant regions.

Thus, antibodies optionally include constant regions or parts thereof,e.g., human antibody constant regions or parts thereof. For example, aVL domain may be attached at its C-terminal end to antibody light chainkappa or lambda constant domains. Similarly, an antibody VH domain maybe attached at its C-terminal end to all or part (e.g. a CH1 domain orFc region) of an immunoglobulin heavy chain constant region derived fromany antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotypesub-classes, such as IgG1 or IgG4.

Examples of human heavy chain constant regions are shown in Table S1.

Constant regions of antibodies of the invention may alternatively benon-human constant regions. For example, when antibodies are generatedin transgenic animals (examples of which are described elsewhereherein), chimaeric antibodies may be produced comprising human variableregions and non-human (host animal) constant regions. Some transgenicanimals generate fully human antibodies. Others have been engineered togenerate antibodies comprising chimaeric heavy chains and fully humanlight chains. Where antibodies comprise one or more non-human constantregions, these may be replaced with human constant regions to provideantibodies more suitable for administration to humans as therapeuticcompositions, as their immunogenicity is thereby reduced.

Digestion of antibodies with the enzyme papain, results in two identicalantigen-binding fragments, known also as “Fab” fragments, and a “Fc”fragment, having no antigen-binding activity but having the ability tocrystallize. “Fab” when used herein refers to a fragment of an antibodythat includes one constant and one variable domain of each of the heavyand light chains. The term “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain, includingnative-sequence Fc regions and variant Fc regions. The “Fc fragment”refers to the carboxy-terminal portions of both H chains held togetherby disulfides. The effector functions of antibodies are determined bysequences in the Fc region, the region which is also recognised by Fcreceptors (FcR) found on certain types of cells. Digestion of antibodieswith the enzyme pepsin, results in the a F(ab′)2 fragment in which thetwo arms of the antibody molecule remain linked and comprise two-antigenbinding sites. The F(ab′)2 fragment has the ability to crosslinkantigen.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites. This regionconsists of a dimer of one heavy and one light chain variable domain intight, non-covalent or covalent association. It is in this configurationthat the three CDRs of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six CDRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree CDRs specific for an antigen) has the ability to recognise andbind antigen, although at a lower affinity than the entire binding site.

Antibodies disclosed herein may be modified to increase or decreaseserum half-life. In one embodiment, one or more of the followingmutations: T252L, T254S or T256F are introduced to increase biologicalhalf-life of the antibody. Biological half-life can also be increased byaltering the heavy chain constant region CH₁ domain or CL region tocontain a salvage receptor binding epitope taken from two loops of a CH₂domain of an Fc region of an IgG, as described in U.S. Pat. Nos.5,869,046 and 6,121,022, the modifications described therein areincorporated herein by reference. In another embodiment, the Fc hingeregion of an antibody or antigen-binding fragment of the invention ismutated to decrease the biological half-life of the antibody orfragment. One or more amino acid mutations are introduced into theCH₂—CH₃ domain interface region of the Fc-hinge fragment such that theantibody or fragment has impaired Staphylococcyl protein A (SpA) bindingrelative to native Fc-hinge domain SpA binding. Other methods ofincreasing serum half-life are known to those skilled in the art. Thus,in one embodiment, the antibody or fragment is PEGylated. In anotherembodiment, the antibody or fragment is fused to an albumin-bidingdomain, e.g. an albumin binding single domain antibody (dAb). In anotherembodiment, the antibody or fragment is PASylated (i.e. genetic fusionof polypeptide sequences composed of PAS (XL-Protein GmbH) which formsuncharged random coil structures with large hydrodynamic volume). Inanother embodiment, the antibody or fragment is XTENylatee/rPEGylated(i.e. genetic fusion of non-exact repeat peptide sequence (Amunix,Versartis) to the therapeutic peptide). In another embodiment, theantibody or fragment is ELPylated (i.e. genetic fusion to ELP repeatsequence (PhaseBio)). These various half-life extending fusions aredescribed in more detail in Strohl, BioDrugs (2015) 29:215-239, whichfusions, e.g. in Tables 2 and 6, are incorporated herein by reference.

The antibody may have a modified constant region which increasesstabililty. Thus, in one embodiment, the heavy chain constant regioncomprises a Ser228Pro mutation. In another embodiment, the antibodiesand fragments disclosed herein comprise a heavy chain hinge region thathas been modified to alter the number of cysteine residues. Thismodification can be used to facilitate assembly of the light and heavychains or to increase or decrease the stability of the antibody.

Fc Effector Functions, ADCC, ADCP and CDC

As discussed above, anti-ICOS antibodies can be provided in variousisotypes and with different constant regions. Examples of human IgGantibody heavy chain constant region sequences are shown in Table S1.The Fc region of the antibody primarily determines its effector functionin terms of Fc binding, antibody-dependent cell-mediated cytotoxicity(ADCC) activity, complement dependent cytotoxicity (CDC) activity andantibody-dependent cell phagocytosis (ADCP) activity. These “cellulareffector functions”, as distinct from effector T cell function, involverecruitment of cells bearing Fc receptors to the site of the targetcells, resulting in killing of the antibody-bound cell. In addition toADCC and CDC, the ADCP mechanism [19] represents a means of depletingantibody-bound T cells, and thus targeting high ICOS expressing TRegsfor deletion.

Cellular effector functions ADCC, ADCP and/or CDC may also be exhibitedby antibodies lacking Fc regions. Antibodies may comprise multipledifferent antigen-binding sites, one directed to ICOS and anotherdirected to a target molecule where engagement of that target moleculeinduces ADCC, ADCP and/or CDC, e.g., an antibody comprising two scFvregions joined by a linker, where one scFv can engage an effector cell.

An antibody according to the present invention may be one that exhibitsADCC, ADCP and/or CDC. Alternatively, an antibody according to thepresent invention may lack ADCC, ADCP and/or CDC activity. In eithercase, an antibody according to the present invention may comprise, ormay optionally lack, an Fc region that binds to one or more types of Fcreceptor. Use of different antibody formats, and the presence or absenceof FcR binding and cellular effector functions, allow the antibody to betailored for use in particular therapeutic purposes as discussedelsewhere herein.

A suitable antibody format for some therapeutic applications employs awild-type human IgG1 constant region. A constant region may be aneffector-enabled IgG1 constant region, optionally having ADCC and/or CDCand/or ADCP activity. A suitable wild type human IgG1 contant regionsequence is SEQ ID NO: 340 (IGHG1*01). Further examples of human IgG1constant regions are shown in Table S1.

For testing of candidate therapeutic antibodies in mouse models of humandisease, an effector positive mouse constant region, such as mouse IgG2a(mIgG2a), may be included instead of an effector positive human constantregion.

A constant region may be engineered for enhanced ADCC and/or CDC and/orADCP.

The potency of Fc-mediated effects may be enhanced by engineering the Fcdomain by various established techniques. Such methods increase theaffinity for certain Fc-receptors, thus creating potential diverseprofiles of activation enhancement. This can achieved by modification ofone or several amino acid residues [20]. Human IgG1 constant regionscontaining specific mutations or altered glycosylation on residue Asn297(e.g., N297Q, EU index numbering) have been shown to enhance binding toFc receptors. Example mutations are one or more of the residues selectedfrom 239, 332 and 330 for human IgG1 constant regions (or the equivalentpositions in other IgG isotypes). An antibody may thus comprise a humanIgG1 constant region having one or more mutations independently selectedfrom N297Q, S239D, I332E and A330L (EU index numbering). A triplemutation (M252Y/S254T/T256E) may be used to enhance binding to FcRn, andother mutations affecting FcRn binding are discussed in Table 2 of [21],any of which may be employed in the present invention.

Increased affinity for Fc receptors can also be achieved by altering thenatural glycosylation profile of the Fc domain by, for example,generating under fucosylated or defucosylated variants [22].Non-fucosylated antibodies harbour a tri-mannosyl core structure ofcomplex-type N-glycans of Fc without fucose residue. Theseglycoengineered antibodies that lack core fucose residue from the FcN-glycans may exhibit stronger ADCC than fucosylated equivalents due toenhancement of FcγRIIIa binding capacity. For example, to increase ADCC,residues in the hinge region can be altered to increase binding toFc-gamma RIII [23]. Thus, an antibody may comprise a human IgG heavychain constant region that is a variant of a wild-type human IgG heavychain constant region, wherein the variant human IgG heavy chainconstant region binds to human Fcγ receptors selected from the groupconsisting of FcγRIIB and FcγRIIA with higher affinity than the wildtype human IgG heavy chain constant region binds to the human Fcγreceptors. The antibody may comprise a human IgG heavy chain constantregion that is a variant of a wild type human IgG heavy chain constantregion, wherein the variant human IgG heavy chain constant region bindsto human FcγRIIB with higher affinity than the wild type human IgG heavychain constant region binds to human FcγRIIB. The variant human IgGheavy chain constant region can be a variant human IgG1, a variant humanIgG2, or a variant human IgG4 heavy chain constant region. In oneembodiment, the variant human IgG heavy chain constant region comprisesone or more amino acid mutations selected from G236D, P238D, S239D,S267E, L328F, and L328E (EU index numbering system). In anotherembodiment, the variant human IgG heavy chain constant region comprisesa set of amino acid mutations selected from the group consisting of:S267E and L328F; P238D and L328E; P238D and one or more substitutionsselected from the group consisting of E233D, G237D, H268D, P271G, andA330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E;S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F(EU index numbering system). The enhancement of CDC may be achieved byamino acid changes that increase affinity for C1q, the first componentof the classic complement activation cascade [24]. Another approach isto create a chimeric Fc domain created from human IgG1 and human IgG3segments that exploit the higher affinity of IgG3 for C1q [25].Antibodies of the present invention may comprise mutated amino acids atresidues 329, 331 and/or 322 to alter the C1q binding and/or reduced orabolished CDC activity. In another embodiment, the antibodies orantibody fragments disclosed herein may contain Fc regions withmodifications at residues 231 and 239, whereby the amino acids arereplaced to alter the ability of the antibody to fix complement. In oneembodiment, the antibody or fragment has a constant region comprisingone or more mutations selected from E345K, E430G, R344D and D356R, inparticular a double mutation comprising R344D and D356R (EU indexnumbering system).

WO2008/137915 described anti-ICOS antibodies with modified Fc regionshaving enhanced effector function. The antibodies were reported tomediate enhanced ADCC activity as compared to the level of ADCC activitymediated by a parent antibody comprising the VH and VK domains and awild type Fc region. Antibodies according to the present invention mayemploy such variant Fc regions having effector function as describedtherein.

ADCC activity of an antibody may be determined in an assay as describedherein. ADCC activity of an anti-ICOS antibody may be determined invitro using an ICOS positive T cell line as described in Example 10.ADCC activity of an anti-PD-L1 antibody may be determined in vitro in anADCC assay using PD-L1 expressing cells.

For certain applications (such as in the context of vaccination) it maybe preferred to use antibodies without Fc effector function. Antibodiesmay be provided without a constant region, or without an Fcregion—examples of such antibody formats are described elsewhere herein.Alternatively, an antibody may have a constant region which is effectornull. An antibody may have a heavy chain constant region that does notbind Fcγ receptors, for example the constant region may comprise aLeu235Glu mutation (i.e., where the wild type leucine residue is mutatedto a glutamic acid residue). Another optional mutation for a heavy chainconstant region is Ser228Pro, which increases stability. A heavy chainconstant region may be an IgG4 comprising both the Leu235Glu mutationand the Ser228Pro mutation. This “IgG4-PE” heavy chain constant regionis effector null.

An alternative effector null human constant region is a disabled IgG1. Adisabled IgG1 heavy chain constant region may contain alanine atposition 235 and/or 237 (EU index numbering), e.g., it may be a IgG1*01sequence comprising the L235A and/or G237A mutations (“LAGA”).

A variant human IgG heavy chain constant region may comprise one or moreamino acid mutations that reduce the affinity of the IgG for humanFcγRIIIA, human FcγRIIA, or human FcγRI. In one embodiment, the FcγRIIBis expressed on a cell selected from the group consisting ofmacrophages, monocytes, B-cells, dendritic cells, endothelial cells, andactivated T-cells. In one embodiment, the variant human IgG heavy chainconstant region comprises one or more of the following amino acidmutations G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V3051,A330L, I332E, E333A, K334A, A339T, and P396L (EU index numberingsystem). In one embodiment, the variant human IgG heavy chain constantregion comprises a set of amino acid mutations selected from the groupconsisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T;S239D and I332E; S239D, A330L, and I332E; S298A, E333A, and K334A;G236A, S239D, and I332E; and F243L, R292P, Y300L, V3051, and P396L (EUindex numbering system). In one embodiment, the variant human IgG heavychain constant region comprises a S239D, A330L, or I332E amino acidmutations (EU index numbering system). In one embodiment, the varianthuman IgG heavy chain constant region comprises an S239D and I332E aminoacid mutations (EU index numbering system). In one embodiment, thevariant human IgG heavy chain constant region is a variant human IgG1heavy chain constant region comprising the S239D and I332E amino acidmutations (EU index numbering system). In one embodiment, the antibodyor fragment comprises an afucosylated Fc region. In another embodiment,the antibody or fragment thereof is defucosylated. In anotherembodiment, the antibody or fragment is under fucosylated.

An antibody may have a heavy chain constant region that binds one ormore types of Fc receptor but does not induce cellular effectorfunctions, i.e., does not mediate ADCC, CDC or ADCP activity. Such aconstant region may be unable to bind the particular Fc receptor(s)responsible for triggering ADCC, CDC or ADCP activity.

Generating and Modifying Antibodies

Methods for identifying and preparing antibodies are well known.Antibodies may be generated using transgenic mice (eg, the Kymouse™,Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse® or MeMo Mouse®), rats(e.g., the Omnirat®), camelids, sharks, rabbits, chickens or othernon-human animals immunised with ICOS or a fragment thereof or asynthetic peptide comprising an ICOS sequence motif of interest,followed optionally by humanisation of the constant regions and/orvariable regions to produce human or humanised antibodies. In anexample, display technologies can be used, such as yeast, phage orribosome display, as will be apparent to the skilled person. Standardaffinity maturation, e.g., using a display technology, can be performedin a further step after isolation of an antibody lead from a transgenicanimal, phage display library or other library. Representative examplesof suitable technologies are described in US20120093818 (Amgen, Inc),which is incorporated by reference herein in its entirety, eg, themethods set out in paragraphs [0309] to [0346].

Immunisation of an ICOS knock out non-human animal with human ICOSantigen facilitates the generation of antibodies that recognise bothhuman and non-human ICOS. As described herein and illustrated in theExamples, an ICOS knock out mouse can be immunised with cells expressinghuman ICOS to stimulate production of antibodies to human and mouse ICOSin the mouse, which can be recovered and tested for binding to humanICOS and to mouse ICOS. Cross-reactive antibodies can thus be selected,which may be screened for other desirable properties as describedherein. Methods of generating antibodies to an antigen (e.g., a humanantigen), through immunisation of animals with the antigen whereexpression of the endogenous antigen (e.g, endogenous mouse antigen) hasbeen knocked-out in the animal, may be performed in animals capable ofgenerating antibodies comprising human variable domains. The genomes ofsuch animals can be engineered to comprise a human or humanisedimmunoglobulin locus encoding human variable region gene segments, andoptionally an endogenous constant region or a human constant region.Recombination of the human variable region gene segments generates humanantibodies, which may have either a non-human or human constant region.Non-human constant regions may subsequently be replaced by humanconstant regions where the antibody is intended for in vivo use inhumans. Such methods and knock-out transgenic animals are described inWO2013/061078.

Generally, a Kymouse™, VELOCIMMUNE® or other mouse or rat (optionally anICOS knock out mouse or rat, as noted) can be challenged with theantigen of interest, and lymphatic cells (such as B-cells) are recoveredfrom the mice that express antibodies. The lymphatic cells may be fusedwith a myeloma cell line to prepare immortal hybridoma cell lines, andsuch hybridoma cell lines are screened and selected to identifyhybridoma cell lines that produce antibodies specific to the antigen ofinterest. DNA encoding the variable regions of the heavy chain and lightchain may be isolated and linked to desirable isotypic constant regionsof the heavy chain and light chain. Such an antibody protein may beproduced in a cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimaeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimaeric antibodies are isolated having ahuman variable region and a mouse constant region. The antibodies arecharacterised and selected for desirable characteristics, includingaffinity, selectivity, agonism, T-cell dependent killing, neutralisingpotency, epitope, etc. The mouse constant regions are optionallyreplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4 (for example, SEQ ID NO: 751, 752, 753 in US2011/0065902 (whichis incorporated by reference herein in its entirety). While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

Thus, in a further aspect, the present invention provides a transgenicnon-human mammal having a genome comprising a human or humanisedimmunoglobulin locus, wherein the mammal does not express ICOS. Themammal may for instance be a knock-out mouse or rat, or other laboratoryanimal species. Transgenic mice such as the Kymouse™ contain human heavyand light chain immunoglobulin loci inserted at the correspondingendogenous mouse immunoglobulin loci. A transgenic mammal according tothe present invention may be one that contains such targeted insertions,or it may contain human heavy and light chain immunoglobulin loci orimmunoglobulin genes that are randomly inserted in its genome, insertedat a locus other than the endogenous Ig locus, or provided on anadditional chromosome or chromosomal fragment.

Further aspects of the invention are the use of such non-human mammalsfor producing antibodies to ICOS, and methods of producing antibodies orantibody heavy and/or light chain variable domains in such mammals.

A method of producing an antibody that binds the extracellular domain ofhuman and non-human ICOS may comprise providing a transgenic non-humanmammal having a genome comprising a human or humanised immunoglobulinlocus, wherein the mammal does not express ICOS, and

(a) immunising the mammal with human ICOS antigen (e.g., with cellsexpressing human ICOS or with purified recombinant ICOS protein);

(b) isolating antibodies generated by the mammal;

(c) testing the antibodies for ability to bind human ICOS and non-humanICOS; and

(d) selecting one or more antibodies that binds both human and non-humanICOS.

Testing for ability to bind human ICOS and non-human ICOS may be doneusing surface plasmon resonance, HTRF, FACS or any other methoddescribed herein. Optionally, binding affinities for human and mouseICOS are determined. The affinity, or fold-difference in affinity, ofbinding to human ICOS and mouse ICOS may be determined, and antibodiesdisplaying species cross-reactivity may thus be selected (affinitythresholds and fold-differences that may be used as selection criteriaare exemplified elsewhere herein). Neutralising potency, or folddifference in neutralising potency, of the antibody for inhibiting humanand mouse ICOS ligand binding to the human and mouse ICOS receptorrespectively may also or alternatively be determined as a way to screenfor cross-reactive antibodies, e.g., in an HTRF assay. Again, possiblethresholds and fold-differences that may be used as selection criteriaare exemplified elsewhere herein.

The method may comprise testing the antibodies for ability to bindnon-human ICOS from the same species or from a different species as theimmunised mammal. Thus, where the transgenic mammal is a mouse (e.g., aKymouse™), antibodies may be tested for ability to bind mouse ICOS.Where the transgenic mammal is a rat, antibodies may be tested forability to bind rat ICOS. However, it may be equally useful to determinecross-reactivity of an isolated antibody for non-human ICOS of anotherspecies. Thus, antibodies generated in goats may be tested for bindingto rat or mouse ICOS. Optionally, binding to goat ICOS may be determinedinstead or additionally.

In other embodiments, the transgenic non-human mammal may be immunisedwith non-human ICOS, optionally ICOS of the same mammalian species(e.g., an ICOS knock-out mouse may be immunised with mouse ICOS) insteadof human ICOS. Affinity of isolated antibodies for binding to human ICOSand non-human ICOS is then determined in the same way, and antibodiesthat bind both human and non-human ICOS are selected.

Nucleic acid encoding an antibody heavy chain variable domain and/or anantibody light chain variable domain of a selected antibody may beisolated. Such nucleic acid may encode the full antibody heavy chainand/or light chain, or the variable domain(s) without associatedconstant region(s). As noted, encoding nucleotide sequences may beobtained directly from antibody-producing cells of a mouse, or B cellsmay be immortalised or fused to generate hybridomas expressing theantibody, and encoding nucleic acid obtained from such cells.Optionally, nucleic acid encoding the variable domain(s) is thenconjugated to a nucleotide sequence encoding a human heavy chainconstant region and/or human light chain constant region, to providenucleic acid encoding a human antibody heavy chain and/or human antibodylight chain, e.g., encoding an antibody comprising both the heavy andlight chain. As described elsewhere herein, this step is particularlyuseful where the immunised mammal produces chimaeric antibodies withnon-human constant regions, which are preferably replaced with humanconstant regions to generate an antibody that will be less immunogenicwhen administered to humans as a medicament. Provision of particularhuman isotype constant regions is also significant for determining theeffector function of the antibody, and a number of suitable heavy chainconstant regions are discussed herein.

Other alterations to nucleic acid encoding the antibody heavy and/orlight chain variable domain may be performed, such as mutation ofresidues and generation of variants, as described herein.

The isolated (optionally mutated) nucleic acid may be introduced intohost cells, e.g., CHO cells as discussed. Host cells are then culturedunder conditions for expression of the antibody, or of the antibodyheavy and/or light chain variable domain, in any desired antibodyformat. Some possible antibody formats are described herein, e.g., wholeimmunoglobulins, antigen-binding fragments, and other designs.

Variable domain amino acid sequence variants of any of the VH and VLdomains or CDRs whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.

There are many reasons why it may be desirable to create variants, whichinclude optimising the antibody sequence for large-scale manufacturing,facilitating purification, enhancing stability or improving suitabilityfor inclusion in a desired pharmaceutical formulation. Proteinengineering work can be performed at one or more target residues in theantibody sequence, e.g., to substituting one amino acid with analternative amino acid (optionally, generating variants containing allnaturally occurring amino acids at this position, with the possibleexception of Cys and Met), and monitoring the impact on function andexpression to determine the best substitution. It is in some instancesundesirable to substitute a residue with Cys or Met, or to introducethese residues into a sequence, as to do so may generate difficulties inmanufacturing—for instance through the formation of new intramolecularor intermolecular cysteine-cysteine bonds. Where a lead candidate hasbeen selected and is being optimised for manufacturing and clinicaldevelopment, it will generally be desirable to change itsantigen-binding properties as little as possible, or at least to retainthe affinity and potency of the parent molecule. However, variants mayalso be generated in order to modulate key antibody characteristics suchas affinity, cross-reactivity or neutralising potency.

An antibody may comprise a set of H and/or L CDRs of any of thedisclosed antibodies with one or more amino acid mutations within thedisclosed set of H and/or L CDRs. The mutation may be an amino acidsubstitution, deletion or insertion. Thus for example there may be oneor more amino acid substitutions within the disclosed set of H and/or LCDRs. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or2 mutations e.g. substitutions, within the set of H and/or L CDRs. Forexample, there may be up to 6, 5, 4, 3 or 2 mutations, e.g.substitutions, in HCDR3 and/or there may be up to 6, 5, 4, 3, or 2mutations, e.g. substitutions, in LCDR3. An antibody may comprise theset of HCDRs, LCDRs or a set of 6 (H and L) CDRs shown for any STIMantibody herein or may comprise that set of CDRs with one or twoconservative substitutions.

One or more amino acid mutations may optionally be made in frameworkregions of an antibody VH or VL domain disclosed herein. For example,one or more residues that differ from the corresponding human germlinesegment sequence may be reverted to germline. Human germline genesegment sequences corresponding to VH and VL domains of exampleanti-ICOS antibodies are indicated in Table E12-1, Table E12-2 and TableE12-3, and alignments of antibody VH and VL domains to correspondinggermline sequences are shown in the drawings.

An antibody may comprise a VH domain that has at least 60, 70, 80, 85,90, 95, 98 or 99 amino acid sequence identity with a VH domain of any ofthe antibodies shown in the appended sequence listing, and/or comprisinga VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% aminoacid sequence identity with a VL domain of any of those antibodies.Algorithms that can be used to calculate % identity of two amino acidsequences include e.g. BLAST, FASTA, or the Smith-Waterman algorithm,e.g. employing default parameters. Particular variants may include oneor more amino acid sequence alterations (addition, deletion,substitution and/or insertion of an amino acid residue).

Alterations may be made in one or more framework regions and/or one ormore CDRs. Variants are optionally provided by CDR mutagenesis. Thealterations normally do not result in loss of function, so an antibodycomprising a thus-altered amino acid sequence may retain an ability tobind ICOS. It may retain the same quantitative binding ability as anantibody in which the alteration is not made, e.g. as measured in anassay described herein. The antibody comprising a thus-altered aminoacid sequence may have an improved ability to bind ICOS.

Alteration may comprise replacing one or more amino acid residue with anon-naturally occurring or non-standard amino acid, modifying one ormore amino acid residue into a non-naturally occurring or non-standardform, or inserting one or more non-naturally occurring or non-standardamino acid into the sequence. Examples of numbers and locations ofalterations in sequences of the invention are described elsewhereherein. Naturally occurring amino acids include the 20 “standard”L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C,K, R, H, D, E by their standard single-letter codes. Non-standard aminoacids include any other residue that may be incorporated into apolypeptide backbone or result from modification of an existing aminoacid residue. Non-standard amino acids may be naturally occurring ornon-naturally occurring.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from a parent polypeptide or nucleic acid by one or moreamino acid or nucleic acid deletions, substitutions or additions, yetretains one or more specific functions or biological activities of theparent molecule. Amino acid substitutions include alterations in whichan amino acid is replaced with a different naturally-occurring aminoacid residue. Such substitutions may be classified as “conservative”, inwhich case an amino acid residue contained in a polypeptide is replacedwith another naturally occurring amino acid of similar character eitherin relation to polarity, side chain functionality or size. Suchconservative substitutions are well known in the art. Substitutionsencompassed by the present invention may also be “non-conservative”, inwhich an amino acid residue which is present in a peptide is substitutedwith an amino acid having different properties, such asnaturally-occurring amino acid from a different group (e.g.,substituting a charged or hydrophobic amino; acid with alanine), oralternatively, in which a naturally-occurring amino acid is substitutedwith a non-conventional amino acid. In some embodiments amino acidsubstitutions are conservative. Also encompassed within the term variantwhen used with reference to a polynucleotide or polypeptide, refers to apolynucleotide or polypeptide that can vary in primary, secondary, ortertiary structure, as compared to a reference polynucleotide orpolypeptide, respectively (e.g., as compared to a wild-typepolynucleotide or polypeptide).

In some aspects, one can use “synthetic variants”, “recombinantvariants”, or “chemically modified” polynucleotide variants orpolypeptide variants isolated or generated using methods well known inthe art. “Modified variants” can include conservative ornon-conservative amino acid changes, as described below. Polynucleotidechanges can result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence. Some aspects use include insertion variants, deletion variantsor substituted variants with substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. The term “conservativesubstitution,” when describing a polypeptide, refers to a change in theamino acid composition of the polypeptide that does not substantiallyalter the polypeptide's activity. For example, a conservativesubstitution refers to substituting an amino acid residue for adifferent amino acid residue that has similar chemical properties (e.g.,acidic, basic, positively or negatively charged, polar or nonpolar,etc.). Conservative amino acid substitutions include replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, ora threonine with a serine. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. For example,the following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

One can select the amino acid that will substitute an existing aminoacid based on the location of the existing amino acid, including itsexposure to solvents (i.e., if the amino acid is exposed to solvents oris present on the outer surface of the peptide or polypeptide ascompared to internally localized amino acids not exposed to solvents).Selection of such conservative amino acid substitutions are well knownin the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999,217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S.French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one canselect conservative amino acid substitutions suitable for amino acids onthe exterior of a protein or peptide (i.e. amino acids exposed to asolvent), for example, but not limited to, the following substitutionscan be used: substitution of Y with F, T with S or K, P with A, E with Dor Q, N with D or G, R with K, G with N or A, T with S or K, D with N orE, I with L or V, F with Y, S with T or A, R with K, G with N or A, Kwith R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

The invention includes methods of producing antibodies containing VHand/or VL domain variants of the antibody VH and/or VL domains shown inthe appended sequence listing. Such antibodies may be produced by amethod comprising

(i) providing, by way of addition, deletion, substitution or insertionof one or more amino acids in the amino acid sequence of a parentantibody VH domain, an antibody VH domain that is an amino acid sequencevariant of the parent antibody VH domain,

wherein the parent antibody VH domain is the VH domain of any ofantibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 and STIM009 or a VH domain comprising theheavy chain complementarity determining regions of any of thoseantibodies,

(ii) optionally combining the VH domain thus provided with a VL domain,to provide a VH/VL combination, and

(iii) testing the VH domain or VH/VL domain combination thus provided toidentify an antibody with one or more desired characteristics.

Desired characteristics include binding to human ICOS, binding to mouseICOS, and binding to other non-human ICOS such as cynomolgus ICOS.Antibodies with comparable or higher affinity for human and/or mouseICOS may be identified. Other desired characteristics include increasingeffector T cell function indirectly, via depletion of immunosuppressiveTRegs, or directly, via ICOS signalling activation on T effector cells.Identifying an antibody with a desired characteristic may compriseidentifying an antibody with a functional attribute described herein,such as its affinity, cross-reactivity, specificity, ICOS receptoragonism, neutralising potency and/or promotion of T cell dependentkilling, any of which may be determined in assays as described herein.

When VL domains are included in the method, the VL domain may be a VLdomain of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 or STIM009, or may be a variant provided byway of addition, deletion, substitution or insertion of one or moreamino acids in the amino acid sequence of a parent VL domain, whereinthe parent VL domain is the VL domain of any of STIM001, STIM002,STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 andSTIM009 or a VL domain comprising the light chain complementaritydetermining regions of any of those antibodies.

Methods of generating variant antibodies may optionally compriseproducing copies of the antibody or VH/VL domain combination. Methodsmay further comprise expressing the resultant antibody. It is possibleto produce nucleotide sequences corresponding to a desired antibody VHand/or VL domain, optionally in one or more expression vectors. Suitablemethods of expression, including recombinant expression in host cells,are set out in detail herein.

Encoding Nucleic Acids and Methods of Expression

Isolated nucleic acid may be provided, encoding antibodies according tothe present invention. Nucleic acid may be DNA and/or RNA. Genomic DNA,cDNA, mRNA or other RNA, of synthetic origin, or any combination thereofcan encode an antibody.

The present invention provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above. Exemplary nucleotide sequences are includedin the sequence listing. Reference to a nucleotide sequence as set outherein encompasses a DNA molecule with the specified sequence, andencompasses a RNA molecule with the specified sequence in which U issubstituted for T, unless context requires otherwise.

The present invention also provides a recombinant host cell thatcomprises one or more nucleic acids encoding the antibody. Methods ofproducing the encoded antibody may comprise expression from the nucleicacid, e.g., by culturing recombinant host cells containing the nucleicacid. The antibody may thus be obtained, and may be isolated and/orpurified using any suitable technique, then used as appropriate. Amethod of production may comprise formulating the product into acomposition including at least one additional component, such as apharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals.

The expression of antibodies and antibody fragments in prokaryotic cellsis well established in the art. A common bacterial host is E. coli.Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production. Mammalian cell linesavailable in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, humanembryonic kidney cells, human embryonic retina cells and many others.

Vectors may contain appropriate regulatory sequences, including promotersequences, terminator sequences, polyadenylation sequences, enhancersequences, marker genes and other sequences as appropriate. Nucleic acidencoding an antibody can be introduced into a host cell. Nucleic acidcan be introduced to eukaryotic cells by various methods, includingcalcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or, for insect cells, baculovirus.Introducing nucleic acid in the host cell, in particular a eukaryoticcell may use a viral or a plasmid based system. The plasmid system maybe maintained episomally or may be incorporated into the host cell orinto an artificial chromosome. Incorporation may be either by random ortargeted integration of one or more copies at single or multiple loci.For bacterial cells, suitable techniques include calcium chloridetransformation, electroporation and transfection using bacteriophage.The introduction may be followed by expressing the nucleic acid, e.g.,by culturing host cells under conditions for expression of the gene,then optionally isolating or purifying the antibody.

Nucleic acid of the invention may be integrated into the genome (e.g.chromosome) of the host cell. Integration may be promoted by inclusionof sequences that promote recombination with the genome, in accordancewith standard techniques.

The present invention also provides a method that comprises usingnucleic acid described herein in an expression system in order toexpress an antibody.

Therapeutic Use

An antibody described herein may be used in a method of treatment of thehuman or animal body by therapy. The antibodies find use in increasingeffector T cell response, which is of benefit for a range of diseases orconditions, including treating cancers or solid tumours and in thecontext of vaccination. Increased Teff response may be achieved using anantibody that modulates the balance or ratio between Teffs and Tregs infavour of Teff activity.

Anti-ICOS antibodies may be used for depleting regulatory T cells and/orincreasing effector T cell response in a patient, and may beadministered to a patient to treat a disease or condition amenable totherapy by depleting regulatory T cells and/or increasing effector Tcell response.

An antibody of the present invention, or a composition comprising suchan antibody molecule or its encoding nucleic acid, may be used orprovided for use in any such method. Use of the antibody, or of acomposition comprising it or its encoding nucleic acid, for themanufacture of a medicament for use in any such method is alsoenvisaged. The method typically comprises administering the antibody orcomposition to a mammal. Suitable formulations and methods ofadministration are described elsewhere herein.

One envisaged therapeutic use of the antibodies is treatment of cancer.The cancer may be a solid tumour, e.g., renal cell cancer (optionallyrenal cell carcinoma, e.g., clear cell renal cell carcinoma), head andneck cancer, melanoma (optionally malignant melanoma), non-small celllung cancer (e.g., adenocarcinoma), bladder cancer, ovarian cancer,cervical cancer, gastric cancer, liver cancer, pancreatic cancer, breastcancer, testicular germ cell carcinoma, or the metastases of a solidtumour such as those listed, or it may be a liquid haematological tumoure.g., lymphoma (such as Hodgkin's lymphoma or Non-Hodgkin's lymphoma,e.g., diffuse large B-cell lymphoma, DLBCL) or leukaemia (e.g., acutemyeloid leukaemia). An anti-ICOS antibody may enhance tumour clearancein melanoma, head and neck cancer and non-small cell lung cancer andother cancers with a moderate to high mutational load [26]. By enhancingpatients' immune response to their neoplastic lesions, immunotherapyusing an anti-ICOS antibody offers the prospect of durable cures orlong-term remissions, potentially even in the context of late stagedisease.

Cancers are a diverse group of diseases, but anti-ICOS antibodies offerthe possibility of treating a range of different cancers by exploitingthe patient's own immune system, which has the potential to kill anycancer cell through recognition of mutant or overexpressed epitopes thatdistinguish cancer cells from normal tissue. By modulating the Teff/Tregbalance, anti-ICOS antibodies can enable and/or promote immunerecognition and killing of cancer cells. While anti-ICOS antibodies aretherefore useful therapeutic agents for a wide variety of cancers, thereare particular categories of cancers for which anti-ICOS therapy isespecially suited and/or where anti-ICOS therapy can be effective whenother therapeutic agents are not.

One such group is cancer that is positive for expression of ICOS ligand.Cancer cells may acquire expression of ICOS ligand, as has beendescribed for melanoma [27]. Expression of ICOS ligand may provide thecells with a selective advantage as the surface-expressed ligand bindsICOS on Tregs, promoting the expansion and activation of the Tregs andthereby suppressing the immune response against the cancer. Cancer cellsexpressing ICOS ligand may depend for their survival on this suppressionof the immune system by Tregs, and would thus be vulnerable to treatmentwith anti-ICOS antibodies that target the Tregs. This applies also tocancers derived from cells that naturally express ICOS ligand. Continuedexpression of ICOS ligand by these cells again provides a survivaladvantage through immune suppression. A cancer expressing ICOS ligandmay be derived from antigen-presenting cells such as B cells, dendriticcells and monocytes and may be a liquid haematological tumour such asthose mentioned herein. Interestingly it has been shown that these typesof cancer are also high in ICOS and FOXP3 expression (TCGA data)—seeExample 25. Example 20 herein demonstrates efficacy of exemplaryanti-ICOS antibodies in treating tumours derived from cancerous B cells(A20 syngeneic cells) that express ICOS ligand.

Accordingly, anti-ICOS antibodies can be used in methods of treatingcancers that are positive for expression of ICOS ligand. Further, acancer to be treated with anti-ICOS antibody according to the presentinvention may be one that is positive for expression of ICOS and/orFOXP3, and optionally also expresses ICOS ligand.

Patients may undergo testing to determine whether their cancer ispositive for expression of the protein of interest (e.g., ICOS ligand,ICOS and/or FOXP3), for example by taking a test sample (e.g., tumourbiopsy) from the patient and determining expression of the protein ofinterest. Patients whose cancer has been characterised as positive forexpression of one, two or all such proteins of interest are selected fortreatment with anti-ICOS antibody. As discussed elsewhere herein,anti-ICOS antibody may be used as a monotherapy or in combination withone or more other therapeutic agents.

Anti-ICOS antibodies also offer hope to patients whose cancers arerefractory to treatment with antibodies or other drugs directed toimmune checkpoint molecules such as CTLA-4, PD-1, PD-L1, CD137, GITR orCD73. These immunotherapies are effective against some cancers but insome cases a cancer may not respond, or it may become unresponsive tocontinued treatment with the antibody. In common with antibodies toimmune checkpoint inhibitors, anti-ICOS antibodies modulate thepatient's immune system—nevertheless an anti-ICOS antibody may succeedwhere such other antibodies fail. It is shown herein that animalscarrying A20 B cell lymphomas could be treated with anti-ICOS antibodiesto reduce growth of the tumour, shrink the tumour and indeed clear thetumour from the body, whereas treatment with an anti-PD-L1 antibody wasno better than control. The A20 cell line has also been reported to beresistant to anti-CTLA-4 [28].

Accordingly, anti-ICOS antibodies can be used in methods of treatingcancers that are refractory to treatment with one or moreimmunotherapies, such as (any or all of) an anti-CTLA-4 antibody,anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody, anti-GITRantibody, or anti-CD73 antibody. A cancer may be characterised as beingrefractory to treatment with an antibody or other drug if treatment withthat antibody or drug does not significantly reduce growth of thecancer, e.g., if a tumour continues to grow or does not reduce in sizeor if after a response period the tumour re-initiates its growth.Non-response to a therapeutic agent may be determined ex vivo by testinga sample (e.g., tumour biopsy sample) for cancer cell killing or growthinhibition, and/or in the clinical setting by observing (e.g., using animaging technology, including MRI) that a patient treated with thetherapy is not responding to treatment. Patients whose cancer has beencharacterised as refractory to treatment with such an immunotherapy areselected for treatment with anti-ICOS antibody.

Further, anti-ICOS antibodies may be used to treat B-cell derived cancerthat is resistant to treatment with an anti-CD20 antibody. Anti-ICOSantibodies represent a treatment for cancers that fail to respond to, orbecome resistant to, therapy with anti-CD20 antibodies like rituximab.Anti-ICOS antibody may be used as a second-line (or further, oradditional) treatment for such cancers. The anti-CD20 antibody resistantcancer may be a B cell cancer, e.g., B cell lymphoma, such as diffuselarge B cell lymphoma. Resistance of a cancer to anti-CD20 may bedetermined ex vivo by testing a sample (e.g., tumour biopsy sample) forcancer cell killing or growth inhibition by anti-CD20 antibody, and/orin the clinical setting by observing that a patient treated with theanti-CD20 antibody is not responding to treatment. Alternatively, oradditionally, the cancer (e.g., a tumour biopsy sample) may be tested toassess expression of CD20, where an absence or low level of CD20expression indicates loss of sensitivity to anti-CD20 antibody.

Samples obtained from patients may thus be tested to determine surfaceexpression of a protein of interest, for example ICOS ligand, ICOS,FOXP3 and/or a target receptor to which another therapeutic agent (e.g.,anti-receptor antibody) is directed. The target receptor may be CD20 (towhich anti-CD20 antibody therapy such as rituximab is directed), oranother receptor such as PD1, EGFR, HER2 or HER3. Surface expression ofICOS ligand, ICOS, FOXP3 and/or lack or loss of surface expression ofthe target receptor is an indication that the cancer is susceptible toanti-ICOS antibody therapy. Anti-ICOS antibodies can be provided foradministration to a patient whose cancer is characterised by surfaceexpression of ICOS ligand, ICOS, FOXP3 and/or lack or loss of surfaceexpression of a target receptor, optionally where the patient has beenpreviously treated with anti-CTLA4, anti-PD1, anti-PD-L1 or with anantibody to the target receptor and has not responded or has stoppedresponding to treatment with that antibody, as measured for example bycontinued or renewed cancer cell growth, e.g., increase in tumour size.

Any suitable method may be employed to determine whether cancer cellstest positive for surface expression of a protein such as ICOS ligand,CD20 or other target receptors mentioned herein. A typical method isimmunohistochemistry, where a sample of the cells (e.g., a tumour biopsysample) is contacted with an antibody for the protein of interest, andbinding of antibody is detected using a labelled reagent—typically asecond antibody that recognises the Fc region of the first antibody andcarries a detectable label such as a fluorescent marker. A sample may bedeclared to test positive where at least 5% of cells are labelled, asvisualised by cell staining or other detection of the label. Optionallya higher cut-off such as 10% or 25% may be used. The antibody willgenerally be used in excess. Reagent antibodies to the molecules ofinterest are available or may be generated by straightforward methods.To test for ICOS ligand, the antibody MAB1651 is currently availablefrom R&D systems as a mouse IgG that recognises human ICOS ligand. Totest for CD20 expression, rituximab may be used. Detection of mRNAlevels of the ICOS ligand or target receptor of interest is analternative technique [27].

A further indication that a tumour will respond to treatment withanti-ICOS antibody is the presence of Tregs in the tumourmicroenvironment. Activated Tregs are characterised by ICOS-high andFoxp3-high surface expression. The presence of Tregs in a tumour,especially in elevated numbers, provides a further basis on which apatient may be selected for treatment with anti-ICOS antibody. Tregs maybe detected in a tumour biopsy sample ex vivo, for example byimmunohistochemistry (assaying for co-expression of both Foxp3 and ICOS,using antibodies to the target protein followed by detection of labels,as described above) or by single cell dispersion of the sample for usein FACS with labelled antibodies to ICOS and Foxp3. FACS methods areexemplified in Example 17 and Example 18.

The anti-ICOS antibodies may be used for treating cancers associatedwith infectious agents, such as virally-induced cancers. In thiscategory are head and neck squamous cell carcinoma, cervical cancer,Merkel cell carcinoma and many others. Viruses associated with cancerinclude HBV, HCV, HPV (cervical cancer, oropharyngeal cancer), and EBV(Burkitts lymphomas, gastric cancer, Hodgkin's lymphoma, other EBVpositive B cell lymphomas, nasopharyngeal carcinoma and post transplantlymphoproliferative disease). The International Agency for Research onCancer (Monograph 100B) identified the following major cancer sitesassociated with infectious agents:

-   -   Stomach/Gastric: Heliobacter pylori    -   Liver: Hepatitis B virus, hepatitis C virus (HCV), Opisthorchis        viverrini, Clonorchis sinensis    -   Cervix uteri: Human papillomavirus (HPV) with or without HIV    -   Anogenital (penile, vulva, vagina, anus): HPV with or without        HIV    -   Nasopharynx: Epstein-Barr virus (EBV)    -   Oropharynx: HPV with or without tobacco or alcohol consumption    -   Kaposi's sarcoma: Human herpes virus type 8 with or without HIV    -   Non-Hodgkin lymphoma: H. pylori, EBV with or without HIV, HCV,        human T-cell lymphotropic virus type 1    -   Hodgkin's lymphoma: EBV with or without HIV    -   Bladder: Schistosoma haematobium.

Antibodies according to the present invention may be used for treatingcancer associated with or induced by any of these infectious agents,such as the cancers specified above.

Stimulation of effector T cell response can also contribute to immunityagainst infectious disease and/or to recovery from infectious disease ina patient. Thus, an anti-ICOS antibody may be used for treatinginfectious disease by administering the antibody to a patient.

Infectious diseases include those caused by pathogens, e.g., bacterial,fungal, viral or protozoal pathogens, and treatment may be to promoteimmune response in a patient against the pathogen infection. An exampleof a bacterial pathogen is tuberculosis. Examples of viral pathogens arehepatitis B and HIV. Examples of protozoal pathogens are Plasmodiumspecies, which cause malaria, such as P. falciparum.

The antibody may be used for treating infections, e.g., infection by anypathogen mentioned herein. Infection may be persistent or chronicinfection. Infection may be localised or systemic. Extended contactbetween a pathogen and the immune system may lead to exhaustion of theimmune system or development of tolerance (manifested for examplethrough increased levels of Tregs, and tipping of the Treg:Teff balancein favour of Tregs) and/or to immune evasion by the pathogen, throughevolution and modification of displayed pathogen antigens. Thesefeatures reflect similar processes that are believed to occur in cancer.Anti-ICOS antibodies present a therapeutic approach to treatinginfection by a pathogen, e.g., chronic infection, through modulation ofthe Treg:Teff ratio in favour of Teff and/or other effects describedherein.

Treatment may be of patients who have been diagnosed as having aninfectious disease or an infection. Alternatively, treatment may bepreventative, and administered to a patient to guard against contractinga disease, e.g., as a vaccine, as described elsewhere herein.

It has also been proposed that an immune response, particularly anIFNγ-dependent systemic immune response, could be beneficial fortreatment of Alzheimer's disease and other CNS pathologies that share aneuroinflammatory component as part [29]. WO2015/136541 proposedtreatment of Alzheimer's disease using an anti-PD-1 antibody. Anti-ICOSantibodies may be used in the treatment of Alzheimer's disease or otherneurodegenerative diseases, optionally in combination with one or moreother immunomodulators (e.g., antibody to PD-1).

Combination Therapy

Treatment with an immunomodulatory antibody such as anti-CTLA4, anti-PD1or anti-PDL1, especially one with Fc effector function, may create anenvironment in which further depletion of ICOS highly expressingimmune-suppressive cells is beneficial. It may be advantageous tocombine an anti-ICOS antibody with such an immunomodulator to enhanceits therapeutic effects.

A patient who has been treated with an immunomodulatory antibody (e.g.,anti-PDL-1, anti-PD-1, anti-CTLA-4) may particularly benefit fromtreatment with an anti-ICOS antibody. One reason for this is that animmunomodulatory antibody may increase the number of ICOS-positive Tregs(e.g., intratumoural Tregs) in the patient. This effect is also observedwith certain other therapeutic agents, such as recombinant IL-2.Anti-ICOS antibody may reduce and/or reverse a surge or rise in ICOS+Tregs (e.g., intratumoural Tregs) resulting from treatment of thepatient with another therapeutic agent. A patient selected for treatmentwith an anti-ICOS antibody may thus be one who has already receivedtreatment with a first therapeutic agent, the first therapeutic agentbeing an antibody (e.g., immunomodulator antibody) or other agent (e.g.,IL-2) that increases the number of ICOS+ Tregs in the patient.

Immunomodulators with which an anti-ICOS antibody may be combinedinclude antibodies to any of: PDL1 (e.g., avelumab), PD-1 (e.g.,pembrolizumab or nivolumab) or CTLA-4 (e.g., ipilimumab ortremelimumab). An anti-ICOS antibody may be combined with pidilizumab.In other embodiments, an anti-ICOS antibody is not administered incombination with anti-CTLA-4 antibody, and/or optionally is administeredin combination with a therapeutic antibody that is not an anti-CTLA-4antibody.

For example, an anti-ICOS antibody may be used in combination therapywith an anti-PDL1 antibody. Preferably, the anti-ICOS antibody is onethat mediates ADCC, ADCP and/or CDC. Preferably, the anti-PDL1 antibodyis one that mediates ADCC, ADCP and/or CDC. An example of suchcombination therapy is administration of an anti-ICOS antibody with ananti-PDL1 antibody wherein both antibodies have effector positiveconstant regions. Thus, the anti-ICOS antibody and the anti-PDL1antibody may both be able to mediate ADCC, CDC and/or ADCP. Fc effectorfunction and selection of constant regions is described in detailelsewhere herein, but as one example an anti-ICOS human IgG1 may becombined with an anti-PD-L1 human IgG1. The anti-ICOS antibody and/orthe anti-PD-L1 antibody may comprise a wild type human IgG1 constantregion. Alternatively, the effector positive constant region of anantibody may be one that is engineered for enhanced effector function,e.g., enhanced CDC, ADCC and/or ADCP. Example antibody constant regions,including wild type human IgG1 sequences and mutations that altereffector function, are discussed in detail elsewhere herein.

Anti-PDL1 antibodies with which an anti-ICOS antibody may be combinedinclude:

-   -   Anti-PDL1 antibody that inhibits binding of PD-1 to PDL1 and/or        inhibits PDL1, optionally as effector positive human IgG1;    -   Anti-PD-1 antibody that inhibits binding of PD-1 to PDL1 and/or        PDL2;    -   Avelumab, a human IgG1 antibody which inhibits PD-1 binding to        PDL-1. See WO2013/079174;    -   Durvalumab (or “MEDI4736”), a variant human IgG1 antibody having        mutations L234A, L235A and 331. See WO2011/066389;    -   Atezolizumab, a variant human IgG1 antibody having mutations        N297A, D356E and L358M. See US2010/0203056;    -   BMS-936559, a human IgG4 antibody comprising mutation S228P. See        WO2007/005874.

Numerous further examples of anti-PD-L1 antibodies are disclosed hereinand others are known in the art. Characterisation data for many of theanti-PD-L1 antibodies mentioned here has been published in U.S. Pat. No.9,567,399 and U.S. Pat. No. 9,617,338, both incorporated by referenceherein. Example anti-PD-L1 antibodies have VH and/or VL domainscomprising the HCDRs and/or LCDRs of any of 1D05, 84G09, 1D05 HC mutant1, 1D05 HC mutant 2, 1D05 HC mutant 3, 1D05 HC mutant 4, 1D05 LC mutant1, 1D05 LC mutant 2, 1D05 LC mutant 3, 411B08, 411C04, 411D07, 385F01,386H03, 389A03, 413D08, 413G05, 413F09, 414B06 or 416E01 as set out inU.S. Pat. No. 9,567,399 or U.S. Pat. No. 9,617,338. The antibody maycomprise the VH and VL domain of any of these antibodies, and mayoptionally comprise a heavy and/or light chain having the heavy and/orlight chain amino acid sequence of any of these antibodies. VH and VLdomains of these anti-PD-L1 antibodies are further described elsewhereherein.

Further example anti-PD-L1 antibodies have VH and/or VL domainscomprising the HCDRs and/or LCDRs of KN-035, CA-170, FAZ-053, M7824,ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504, or of ananti-PD-L1 antibody disclosed in any of WO2017/034916, WO2017/020291,WO2017/020858, WO2017/020801, WO2016/111645, WO2016/197367,WO2016/061142, WO2016/149201, WO2016/000619, WO2016/160792,WO2016/022630, WO2016/007235, WO2015/179654, WO2015/173267,WO2015/181342, WO2015/109124, WO2015/112805, WO2015/061668,WO2014/159562, WO2014/165082, WO2014/100079, WO2014/055897,WO2013/181634, WO2013/173223, WO2013/079174, WO2012/145493,WO2011/066389, WO2010/077634, WO2010/036959, WO2010/089411 andWO2007/005874. The antibody may comprise the VH and VL domain of any ofthese antibodies, and may optionally comprise a heavy and/or light chainhaving the heavy and/or light chain amino acid sequence of any of theseantibodies. The anti-ICOS antibody which is used in combination therapywith anti-PD-L1 may be an antibody of the present invention as disclosedherein. Alternatively, the anti-ICOS antibody may comprise the CDRs of,or a VH and/or VL domain of, an anti-ICOS antibody disclosed in any ofthe following publications:

WO2016154177, US2016304610—for example any of antibodies 7F12, 37A10,35A9, 36E10, 16G10, 37A10S713, 37A10S714, 37A10S715, 37A10S716,37A10S717, 37A10S718, 16G10S71, 16G10S72, 16G10S73, 16G10S83, 35A9S79,35A9S710, or 35A9S89;

WO16120789, US2016215059—for example the antibody known as 422.2 and/orH2L5;

WO14033327, EP2892928, US2015239978—for example the antibody known as314-8 and/or produced from hybridoma CNCM I-4180;

WO12131004, EP2691419, U.S. Pat. No. 9,376,493, US20160264666—forexample the antibody Icos145-1 and/or antibody produced by hybridomaCNCM I-4179;

WO10056804—for example the antibody JMAb 136 or “136”;

WO9915553, EP1017723B1, U.S. Pat. No. 7,259,247, U.S. Pat. No.7,132,099, U.S. Pat. No. 7,125,551, U.S. Pat. No. 7,306,800, U.S. Pat.No. 7,722,872, WO05103086, EP1740617, U.S. Pat. No. 8,318,905, U.S. Pat.No. 8,916,155—for example the antibody MIC-944 or 9F3;WO983821, U.S. Pat. No. 7,932,358B2, US2002156242, EP0984023, EP1502920,U.S. Pat. No. 7,030,225, U.S. Pat. No. 7,045,615, U.S. Pat. No.7,279,560, U.S. Pat. No. 7,226,909, U.S. Pat. No. 7,196,175, U.S. Pat.No. 7,932,358, U.S. Pat. No. 8,389,690, WO02070010, EP1286668,EP1374901, U.S. Pat. No. 7,438,905, U.S. Pat. No. 7,438,905, WO0187981,EP1158004, U.S. Pat. No. 6,803,039, U.S. Pat. No. 7,166,283, U.S. Pat.No. 7,988,965, WO0115732, EP1125585, U.S. Pat. No. 7,465,445, U.S. Pat.No. 7,998,478—for example any JMAb antibody, e.g., any of JMAb-124,JMAb-126, JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb-138,JMAb-139, JMAb-140, JMAb-141, e.g., JMAb136;WO2014/089113—for example antibody 17G9;WO12174338;US2016145344;WO11020024, EP2464661, US2016002336, US2016024211, U.S. Pat. No.8,840,889;U.S. Pat. No. 8,497,244.

The anti-ICOS antibody optionally comprises the CDRs of 37A10S713 asdisclosed in WO2016154177. It may comprise the VH and VL domains of37A10S713, and may optionally have the antibody heavy and light chainsof 37A10S713.

Combination of an anti-ICOS antibody with an immunomodulator may providean increased therapeutic effect compared with monotherapy, and may allowtherapeutic benefit to be achieved with a lower dose of theimmunomodulator(s). Thus, for example, an antibody (e.g., anti-PD-L1antibody, optionally ipilimumab) that is used in combination withanti-ICOS antibody may be dosed at 3 mg/kg rather than a more usual doseof 10 mg/kg. The administration regimen of the anti-PD-L1 or otherantibody may involve intravenous administration over a 90 minute periodevery 3 weeks for a total of 4 doses.

An anti-ICOS antibody may be used to increase the sensitivity of atumour to treatment with an anti-PD-L1 antibody, which may be recognisedas a reduction in the dose at which the anti-PD-L1 antibody exerts atherapeutic benefit. Thus, anti-ICOS antibody may be administered to apatient to reduce the dose of anti-PD-L1 antibody effective to treatcancer or a tumour in the patient. Administration of anti-ICOS antibodymay reduce the recommended or required dosage of anti-PD-L1 antibodyadministration to that patient to, for example, 75%, 50%, 25%, 20%, 10%or less, compared with the dosage when anti-PD-L1 antibody isadministered without anti-ICOS. The patient may be treated byadministration of anti-ICOS antibody and anti-PD-L1 antibody in acombination therapy as described herein.

The benefit of combining anti-PD-L1 with anti-ICOS may extend to areduction in dosage of each agent when compared with its use as amonotherapy. Anti-PD-L1 antibody may be used to reduce the dose at whichanti-ICOS antibody exerts a therapeutic benefit, and thus may beadministered to a patient to reduce the dose of anti-ICOS antibodyeffective to treat cancer or a tumour in the patient. Thus, ananti-PD-L1 antibody may reduce the recommended or required dosage ofanti-ICOS antibody administration to that patient to, for example, 75%,50%, 25%, 20%, 10% or less, compared with the dosage when anti-ICOSantibody is administered without anti-PD-L1. The patient may be treatedby administration of anti-ICOS antibody and anti-PD-L1 antibody in acombination therapy as described herein.

As discussed in Example 22 herein, treatment with anti-PD-L1 antibody,especially antibody with effector positive Fc, appears not to increasethe expression of ICOS on Teff cells. This is advantageous whenadministering such antibodies in combination with effector positiveanti-ICOS antibodies, where an increase in ICOS expression on Teffswould undesirably render these cells more sensitive to depletion by theanti-ICOS antibody. In a combination with anti-PD-L1, anti-ICOS therapymay thus exploit a differential expression of ICOS on Teffs comparedwith Tregs, preferentially targeting the ICOS-high Tregs for depletion.This in turn relieves the suppression of TEffs and has a net effect ofpromoting the effector T cell response in a patient. The effect oftargeting immune checkpoint molecules on expression of ICOS on T cellshas also been studied previously—see Figure S6C in ref. [30](supplementary materials), where treatment with CTLA-4 antibody and/oranti-PD-1 antibody was reported to increase the percentage of CD4+ Tregsexpressing ICOS. The effect of a therapeutic agent on ICOS expression inTregs and Teffs may be a factor in selection of appropriate agents foruse in combination with anti-ICOS antibodies, noting that effect of theanti-ICOS antibody may be enhanced under conditions where there is highdifferential expression of ICOS on Tregs versus Teffs.

As described herein, a single dose of anti-ICOS antibody may besufficient to provide therapeutic effect, especially in combination withother therapeutic agents such as anti-PD-L1 antibody. In tumour therapy,the underlying rationale for this single dose benefit may be that theanti-ICOS antibody mediates its effect, at least in part, by resettingor altering the microenvironment of the tumour sufficiently to renderthe tumour more sensitive to immune attack and/or to the effects ofother immunomodulators such as those mentioned. Tumour microenviromentresetting is triggered through for example depletion of ICOS positivetumour infiltrating T-regs. So, for example, a patient may be treatedwith a single dose of an anti-ICOS antibody followed by one or multipledoses of anti-PD-L1 antibody. Over a period of treatment, for examplesix months or a year, the anti-ICOS antibody may be administered in asingle dose while other agents, e.g., anti-PD-L1 antibody, areoptionally administered multiple times over that treatment period,preferably with at least one such dose being administered subsequent totreatment with the anti-ICOS antibody.

Further examples of combination therapy include combination of anti-ICOSantibody with:

-   -   an antagonist of an adenosine A2A receptor (“A2AR inhibitor”);    -   a CD137 agonist (e.g., agonist antibody);    -   an antagonist of the enzyme indoleamine-2,3 dioxygenase, which        catalyses the breakdown of tryptophan (“IDO inhibitor”). IDO is        an immune checkpoint, activated in dendritic cells and        macrophages, which contributes to immune suppression/tolerance.

Anti-ICOS antibodies may be used in combination therapy with IL-2 (e.g.,recombinant IL-2 such as aldesleukin). The IL-2 may be administered athigh dose (HD). Typical HD IL-2 therapy involves bolus infusion of over500,000 IU/kg, e.g., bolus infusions of 600,000 or 720,000 IU/kg, percycle of therapy, where 10-15 such bolus infusions are given atintervals of between 5-10 hours, e.g., up to 15 bolus infusions every 8hours, and repeating the therapy cycle approximately every 14 to 21 daysfor up to 6 to 8 cycles. HD IL-2 therapy has been successful in treatingtumours, especially melanoma (e.g., metastatic melanoma) and renal cellcarcinoma, but its use is limited to the high toxicity of IL-2 which cancause severe adverse effects.

Treatment with high dose IL-2 has been shown to increase the populationof ICOS-positive Tregs in cancer patients [31]. This increase in ICOS+TRegs following the first cycle of HD IL-2 therapy was reported tocorrelate with worse clinical outcome—the higher the number of ICOS+Tregs, the worse the prognosis. An IL-2 variant F42K has been proposedas an alternative therapy to avoid this undesirable increase in ICOS+Treg cells [32]. However, another approach would be to exploit theincrease in ICOS+ T regs by using an antibody in accordance with thepresent invention as a second-line therapeutic agent.

It may be beneficial to combine IL-2 therapy with anti-ICOS antibodies,capitalising on the ability of anti-ICOS antibodies to target TRegs thathighly express ICOS, inhibiting these cells and improving the prognosisfor patients undergoing IL-2 therapy. Concomitant administration of IL-2and anti-ICOS antibody may increase the response rate while avoiding orreducing adverse events in the treated patient population. Thecombination may permit IL-2 to be used at lower dose compared with IL-2monotherapy, reducing the risk or level of adverse events arising fromthe IL-2 therapy, while retaining or enhancing clinical benefit (e.g.,reduction of tumour growth, clearance of solid tumour and/or reductionof metastasis). In this way, addition of anti-ICOS can improve treatmentof patients who are receiving IL-2, whether high-dose (HD) or low-dose(LD) IL-2.

Accordingly, one aspect of the invention provides a method of treating apatient by administering an anti-ICOS antibody to the patient, whereinthe patient is also treated with IL-2, e.g., HD IL-2. Another aspect ofthe invention is an anti-ICOS antibody for use in treating a patient,wherein the patient is also treated with IL-2, e.g., HD IL-2. Theanti-ICOS antibody may be used as a second-line therapy. Thus, thepatient may be one who has been treated with IL-2, e.g., having receivedat least one cycle of HD IL-2 therapy, and who has an increased level ofICOS+ Tregs. Assays may be performed on samples of cancer cells, e.g.,tumour biopsy samples, using immunohistochemistry or FACS as describedelsewhere herein to detect cells positive for ICOS, Foxp3, ICOSL andoptionally one or more further markers of interest. Methods may comprisedetermining that the patient has an increased level of ICOS+ Tregs(e.g., in peripheral blood, or in a tumour biopsy) following IL-2treatment, where an increased level is indicative that the patient wouldbenefit from treatment with the anti-ICOS antibody. The increase inTregs may be relative to control (untreated) individuals or to thepatient prior to IL-2 therapy. Such patients with elevated Tregsrepresent a group who may not benefit from continued IL-2 treatmentalone, but for whom a combination of anti-ICOS antibody and IL-2therapy, or treatment with anti-ICOS antibody alone, offers therapeuticbenefit. Thus, following a positive determination that the patient hasan increased level of ICOS+ Tregs, anti-ICOS antibody and/or furtherIL-2 therapy may be administered. Treatment with the anti-ICOS antibodymay selectively target and deplete the ICOS+ Tregs relative to other Tcell populations in such patients. This provides a therapeutic effect byrelieving the immunosuppression mediated by these cells and therebyenhancing activity of Teffs against the target cells, e.g., tumour cellsor infected cells.

Combination therapy with anti-ICOS antibodies and IL-2 may be used forany therapeutic indication described herein, and particularly fortreating a tumour, e.g., melanoma such as metastatic melanoma, or renalcell carcinoma. Thus, in one example, the patient treated with ananti-ICOS antibody is one who presents with metastatic melanoma and hasbeen treated with IL-2, e.g., HD IL-2 therapy or LD IL-2 therapy.

In general, where an anti-ICOS antibody is administered to a patient whohas received treatment with a first therapeutic agent (e.g.,immunomodulator antibody) or other agent (e.g., IL-2), the anti-ICOSantibody may be administered after a minimum period of, for example, 24hours, 48 hours, 72 hours, 1 week or 2 weeks following administration ofthe first therapeutic agent. The anti-ICOS antibody may be administeredwithin 2, 3, 4 or 5 weeks after administration of the first therapeuticagent. This does not exclude additional administrations of either agentat any time, although it may be desirable to minimise the number oftreatments administered, for ease of compliance for patients and toreduce costs. Rather, the relative timing of the administrations will beselected to optimise their combined effect, the first therapeutic agentcreating an immunological environment (e.g., elevated ICOS+ Tregs, orantigen release as discussed below) in which the effect of the anti-ICOSantibody is especially advantageous. Thus, sequential administration ofthe first therapeutic agent and then the anti-ICOS antibody may allowtime for the first agent to act, creating in vivo conditions in whichthe anti-ICOS antibody can exhibit its enhanced effect. Variousadministration regimens, including simultaneous or sequentialcombination treatments, are described herein and can be utilised asappropriate. Where the first therapeutic agent is one that increases thenumber of ICOS+ Tregs in the patient, the treatment regimen for thepatient may comprise determining that the patient has an increasednumber of ICOS+ Tregs, and then administering the anti-ICOS antibody.

As noted, use of anti-ICOS antibodies in combination therapy may provideadvantages of reducing the effective dose of the therapeutic agentsand/or countering adverse effects of therapeutic agents that increaseICOS+ Tregs in patients. Yet further therapeutic benefits may beachieved through selecting a first therapeutic agent that causes releaseof antigens from target cells through “immunological cell death”, andadministering the first therapeutic agent in combination with ananti-ICOS antibody. As noted, administration of the anti-ICOS antibodymay sequentially follow administration of the first therapeutic agent,administration of the two agents being separated by a certain timewindow as discussed above.

Immunological cell death is a recognised mode of cell death, contrastingwith apoptosis. It is characterised by release of ATP and HMGB1 from thecell and exposure of calreticulin on the plasma membrane [33, 34].

Immunological cell death in a target tissue or in target cells promotesengulfment of the cell by an antigen-presenting cell, resulting indisplay of antigens from the target cell, which in turn inducesantigen-specific Teff cells. Anti-ICOS antibody may increase themagnitude and/or duration of the Teff response by acting as an agonistof ICOS on the Teff cells. In addition, where the anti-ICOS antibody isFc effector function enabled (e.g., a human IgG1 antibody), theanti-ICOS antibody may cause depletion of antigen-specific Tregs. Thus,through a combination of either or both of these effects, the balancebetween Teff and Treg cells is modulated in favour of enhancing Teffactivity. Combination of an anti-ICOS antibody with a treatment thatinduces immunological cell death in a target tissue or cell type, suchas in a tumour or in cancer cells, thereby promotes an immune responsein the patient against the target tissue or cells, representing a formof vaccination in which the vaccine antigen is generated in vivo.

Accordingly, one aspect of the invention is a method of treating cancerin a patient by in vivo vaccination of the patient against their cancercells. Another aspect of the invention is an anti-ICOS antibody for usein such a method. Anti-ICOS antibodies may be used in a methodcomprising:

-   -   treating the patient with a therapy that causes immunological        cell death of the cancer cells, resulting in presentation of        antigen to antigen-specific effector T cells, and    -   administering an anti-ICOS antibody to the patient, wherein the        anti-ICOS antibody enhances the antigen-specific effector T cell        response against the cancer cells.

Treatments that induce immunological cell death include radiation (e.g.,ionising irradiation of cells using UVC light or γ rays),chemotherapeutic agents (e.g., oxaliplatin, anthracyclines such asdoxorubicin, idarubicin or mitoxantrone, BK channel agonists such asphloretin or pimaric acid, bortezomib, cardiac glycosides,cyclophosphamide, GADD34/PP1 inhibitors with mitomycin, PDT withhypericin, polyinosinic-polycytidylic acid, 5-fluorouracil, gemcitabine,gefitnib, erlotinib, or thapsigargin with cisplatin) and antibodies totumour-associated antigens. The tumour-associated antigen can be anyantigen that is over-expressed by tumour cells relative to non-tumourcells of the same tissue, e.g., HER2, CD20, EGFR. Suitable antibodiesinclude herceptin (anti-HER2), rituximab (anti-CD20), or cetuximab(anti-EGFR).

Thus, it is advantagous to combine an anti-ICOS antibody with one ormore such treatments. Optionally, the anti-ICOS antibody is adminsteredto a patient who has already received such treatment. The anti-ICOSantibody may be administered after a period of, for example, 24 hours,48 hours, 72 hours, 1 week or 2 weeks following the treatment thatinduces immunological cell death, e.g., between 24 to 72 hours after thetreatment. The anti-ICOS antibody may be administered within 2, 3, 4 or5 weeks after the treatment. Other regimens for combination therapy arediscussed elsewhere herein.

While “in vivo vaccination” has been described above, it is alsopossible to treat tumour cells to induce immunological cell death exvivo, after which the cells may be reintroduced to the patient. Ratherthan administering the agent or treatment that induces immunologicalcell death directly to the patient, the treated tumour cells areadministered to the patient. Treatment of the patient may be inaccordance with administration regimens described above.

As already noted, a single dose of an anti-ICOS antibody may besufficient to provide therapeutic benefit. Thus, in the methods oftreatment described herein, the anti-ICOS antibody is optionallyadministered as a single dose. A single dose of anti-ICOS antibody maydeplete Tregs in a patient, with consequent beneficial effects indiseases such as cancer. It has previously been reported that transientablation of Tregs has anti-tumour effects, including reducing tumourprogression, treating established tumours and metastases and extendingsurvival, and that it can enhance the therapeutic effect of tumourirradiation [35]. Administration of a single dose of anti-ICOS mayprovide such Treg depletion, and may be used to enhance the effects ofother therapeutic approaches used in combination, such as radiotherapy.

Antibodies to PD-L1

An antibody to PD-L1 for use in combination with an anti-ICOS antibody,whether as a separate therapeutic agent or in a multispecific antibodyas described herein, may comprise the antigen-binding site of anyanti-PD-L1 antibody. Numerous examples of anti-PD-L1 antibodies aredisclosed herein and others are known in the art. Characterisation datafor many of the anti-PD-L1 antibodies mentioned here has been publishedin U.S. Pat. No. 9,567,399 and U.S. Pat. No. 9,617,338, bothincorporated by reference herein.

1D05 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequenceof Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleicacid sequence of the V_(H) domain is Seq ID No:34. 1D05 has a lightchain variable region (V_(L)) amino acid sequence of Seq ID No:43,comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or SeqID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT)or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq IDNo:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acidsequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq IDNo:524, Seq ID No: 526, Seq ID No:528, Seq ID No: 530, Seq ID No: 532 orSeq ID No: 534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full lengthlight chain amino acid sequence is Seq ID No:45 (light chain nucleicacid sequence Seq ID No:46).

84G09 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:13, comprising the CDRH1 amino acid sequence of Seq ID No:7(IMGT) or Seq ID No:10 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:8 (IMGT) or Seq ID No:11 (Kabat), and the CDRH3 amino acid sequenceof Seq ID No:9 (IMGT) or Seq ID No:12 (Kabat). The heavy chain nucleicacid sequence of the V_(H) domain is Seq ID No:14. 84G09 has a lightchain variable region (V_(L)) amino acid sequence of Seq ID No:23,comprising the CDRL1 amino acid sequence of Seq ID No:17 (IMGT) or SeqID No:20 (Kabat), the CDRL2 amino acid sequence of Seq ID No:18 (IMGT)or Seq ID No:21 (Kabat), and the CDRL3 amino acid sequence of Seq IDNo:19 (IMGT) or Seq ID No:22 (Kabat). The light chain nucleic acidsequence of the V_(L) domain is Seq ID No:24. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq IDNo:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 orSeq ID No:534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:15 (heavy chain nucleic acid sequence Seq ID No:16). A full lengthlight chain amino acid sequence is Seq ID No:25 (light chain nucleicacid sequence Seq ID No:26).

1D05 HC mutant 1 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:47, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 1 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:43, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acidsequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). Thelight chain nucleic acid sequence of the V_(L) domain is Seq ID No:44.The V_(H) domain may be combined with any of the heavy chain constantregion sequences described herein, e.g. Seq ID No:193, Seq ID No:195,Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq IDNo:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, SeqID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length lightchain amino acid sequence is Seq ID No:45 (light chain nucleic acidsequence Seq ID No:46).

1D05 HC mutant 2 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:48, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 2 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:43, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acidsequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). Thelight chain nucleic acid sequence of the V_(L) domain is Seq ID No:44.The V_(H) domain may be combined with any of the heavy chain constantregion sequences described herein, e.g. Seq ID No:193, Seq ID No:195,Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq IDNo:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, SeqID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length lightchain amino acid sequence is Seq ID No:45 (light chain nucleic acidsequence Seq ID No:46).

1D05 HC mutant 3 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:49, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 3 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:43, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acidsequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). Thelight chain nucleic acid sequence of the V_(L) domain is Seq ID No:44.The V_(H) domain may be combined with any of the heavy chain constantregion sequences described herein, e.g. Seq ID No:193, Seq ID No:195,Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq IDNo:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, SeqID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length lightchain amino acid sequence is Seq ID No:45 (light chain nucleic acidsequence Seq ID No:46).

1D05 HC mutant 4 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:342, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 4 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:43, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acidsequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). Thelight chain nucleic acid sequence of the V_(L) domain is Seq ID No:44.The V_(H) domain may be combined with any of the heavy chain constantregion sequences described herein, e.g. Seq ID No:193, Seq ID No:195,Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq IDNo:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, SeqID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may becombined with any of the light chain constant region sequences describedherein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length lightchain amino acid sequence is Seq ID No:45 (light chain nucleic acidsequence Seq ID No:46).

1D05 LC mutant 1 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:33, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). Theheavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34.1D05 LC mutant 1 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:50, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), and the CDRL3 amino acidsequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2sequence of 1D05 LC Mutant 1 is as defined by the Kabat or IMGT systemsfrom the V_(L) sequence of Seq ID No:50. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq IDNo:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 orSeq ID No:534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:35 (heavy chain nucleic acid sequence Seq ID No:36).

1D05 LC mutant 2 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:33, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). Theheavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34.1D05 LC mutant 2 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:51, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acidsequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). TheV_(H) domain may be combined with any of the heavy chain constant regionsequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq IDNo:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, SeqID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530,Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined withany of the light chain constant region sequences described herein, e.g.Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq IDNo:36).

1D05 LC mutant 3 has a heavy chain variable (V_(H)) region amino acidsequence of Seq ID No:33, comprising the CDRH1 amino acid sequence ofSeq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acidsequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). Theheavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34.1D05 LC mutant 3 has a light chain variable region (V_(L)) amino acidsequence of Seq ID No:298, comprising the CDRL1 amino acid sequence ofSeq ID No:37 (IMGT) or Seq ID No:40 (Kabat), and the CDRL3 amino acidsequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2sequence of 1D05 LC Mutant 3 is as defined by the Kabat or IMGT systemsfrom the V_(L) sequence of Seq ID No:298. The light chain nucleic acidsequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq IDNo:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 orSeq ID No:534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full lengthlight chain amino acid sequence is Seq ID No:45 (light chain nucleicacid sequence Seq ID No:46).

411B08 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:58, comprising the CDRH1 amino acid sequence of Seq ID No:52(IMGT) or Seq ID No:55 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:53 (IMGT) or Seq ID No:56 (Kabat), and the CDRH3 amino acid sequenceof Seq ID No:54 (IMGT) or Seq ID No:57 (Kabat). The heavy chain nucleicacid sequence of the V_(H) domain is Seq ID No:59. 411B08 has a lightchain variable region (V_(L)) amino acid sequence of Seq ID No:68,comprising the CDRL1 amino acid sequence of Seq ID No:62 (IMGT) or SeqID No:65 (Kabat), the CDRL2 amino acid sequence of Seq ID No:63 (IMGT)or Seq ID No:66 (Kabat), and the CDRL3 amino acid sequence of Seq IDNo:64 (IMGT) or Seq ID No:67 (Kabat). The light chain nucleic acidsequence of the V_(L) domain is Seq ID No:69. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq IDNo:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 orSeq ID No:534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:60 (heavy chain nucleic acid sequence Seq ID No:61). A full lengthlight chain amino acid sequence is Seq ID No:70 (light chain nucleicacid sequence Seq ID No:71).

411C04 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:78, comprising the CDRH1 amino acid sequence of Seq ID No:72(IMGT) or Seq ID No:75 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:73 (IMGT) or Seq ID No:76 (Kabat), and the CDRH3 amino acid sequenceof Seq ID No:74 (IMGT) or Seq ID No:77 (Kabat). The heavy chain nucleicacid sequence of the V_(H) domain is Seq ID No:79. 411C04 has a lightchain variable region (V_(L)) amino acid sequence of Seq ID No:88,comprising the CDRL1 amino acid sequence of Seq ID No:82 (IMGT) or SeqID No:85 (Kabat), the CDRL2 amino acid sequence of Seq ID No:83 (IMGT)or Seq ID No:86 (Kabat), and the CDRL3 amino acid sequence of Seq IDNo:84 (IMGT) or Seq ID No:87 (Kabat). The light chain nucleic acidsequence of the V_(L) domain is Seq ID No:89. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq IDNo:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 orSeq ID No:534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:80 (heavy chain nucleic acid sequence Seq ID No:81). A full lengthlight chain amino acid sequence is Seq ID No:90 (light chain nucleicacid sequence Seq ID No:91).

411D07 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:98, comprising the CDRH1 amino acid sequence of Seq ID No:92(IMGT) or Seq ID No:95 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:93 (IMGT) or Seq ID No:96 (Kabat), and the CDRH3 amino acid sequenceof Seq ID No:94 (IMGT) or Seq ID No:97 (Kabat). The heavy chain nucleicacid sequence of the V_(H) domain is Seq ID No:99. 411D07 has a lightchain variable region (V_(L)) amino acid sequence of Seq ID No:108,comprising the CDRL1 amino acid sequence of Seq ID No:102 (IMGT) or SeqID No:105 (Kabat), the CDRL2 amino acid sequence of Seq ID No:103 (IMGT)or Seq ID No:106 (Kabat), and the CDRL3 amino acid sequence of Seq IDNo:104 (IMGT) or Seq ID No:107 (Kabat). The light chain nucleic acidsequence of the V_(L) domain is Seq ID No:109. The V_(H) domain may becombined with any of the heavy chain constant region sequences describedherein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199,Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq IDNo:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 orSeq ID No:534. The V_(L) domain may be combined with any of the lightchain constant region sequences described herein, e.g. Seq ID Nos:207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 536 and 538. A full length heavy chain amino acid sequence is SeqID No:100 (heavy chain nucleic acid sequence Seq ID No:101). A fulllength light chain amino acid sequence is Seq ID No: 110 (light chainnucleic acid sequence Seq ID No:111).

385F01 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:118, comprising the CDRH1 amino acid sequence of Seq ID No:112(IMGT) or Seq ID No:115 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:113 (IMGT) or Seq ID No:116 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:114 (IMGT) or Seq ID No:117 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:119. 385F01has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:128, comprising the CDRL1 amino acid sequence of Seq ID No:122 (IMGT)or Seq ID No:125 (Kabat), the CDRL2 amino acid sequence of Seq ID No:123(IMGT) or Seq ID No:126 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:124 (IMGT) or Seq ID No:127 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:129. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:120 (heavy chain nucleic acid sequence Seq IDNo:121). A full length light chain amino acid sequence is Seq ID No:130(light chain nucleic acid sequence Seq ID No:131).

386H03 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:158, comprising the CDRH1 amino acid sequence of Seq ID No:152(IMGT) or Seq ID No:155 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:153 (IMGT) or Seq ID No:156 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:154 (IMGT) or Seq ID No:157 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:159. 386H03has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:168, comprising the CDRL1 amino acid sequence of Seq ID No:162 (IMGT)or Seq ID No:165 (Kabat), the CDRL2 amino acid sequence of Seq ID No:163(IMGT) or Seq ID No:166 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:164 (IMGT) or Seq ID No:167 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:169. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:160 (heavy chain nucleic acid sequence Seq IDNo:161). A full length light chain amino acid sequence is Seq ID No:170(light chain nucleic acid sequence Seq ID No:171).

389A03 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:178, comprising the CDRH1 amino acid sequence of Seq ID No:172(IMGT) or Seq ID No:175 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:173 (IMGT) or Seq ID No:176 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:174 (IMGT) or Seq ID No:177 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:179. 389A03has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:188, comprising the CDRL1 amino acid sequence of Seq ID No:182 (IMGT)or Seq ID No:185 (Kabat), the CDRL2 amino acid sequence of Seq ID No:183(IMGT) or Seq ID No:186 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:184 (IMGT) or Seq ID No:187 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:189. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:180 (heavy chain nucleic acid sequence Seq IDNo:181). A full length light chain amino acid sequence is Seq ID No:190(light chain nucleic acid sequence Seq ID No:191).

413D08 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:138, comprising the CDRH1 amino acid sequence of Seq ID No:132(IMGT) or Seq ID No:135 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:133 (IMGT) or Seq ID No:136 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:134 (IMGT) or Seq ID No:137 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:139. 413D08has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:148, comprising the CDRL1 amino acid sequence of Seq ID No:142 (IMGT)or Seq ID No:145 (Kabat), the CDRL2 amino acid sequence of Seq ID No:143(IMGT) or Seq ID No:146 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:144 (IMGT) or Seq ID No:147 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:149. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No: 140 (heavy chain nucleic acid sequence Seq IDNo:141). A full length light chain amino acid sequence is Seq ID No:150(light chain nucleic acid sequence Seq ID No:151).

413G05 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:244, comprising the CDRH1 amino acid sequence of Seq ID No:238(IMGT) or Seq ID No:241 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:239 (IMGT) or Seq ID No:242 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:240 (IMGT) or Seq ID No:243 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:245. 413G05has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:254, comprising the CDRL1 amino acid sequence of Seq ID No:248 (IMGT)or Seq ID No:251 (Kabat), the CDRL2 amino acid sequence of Seq ID No:249(IMGT) or Seq ID No:252 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:250 (IMGT) or Seq ID No:253 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:255. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:246 (heavy chain nucleic acid sequence Seq IDNo:247). A full length light chain amino acid sequence is Seq ID No:256(light chain nucleic acid sequence Seq ID No:257).

413F09 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:264, comprising the CDRH1 amino acid sequence of Seq ID No:258(IMGT) or Seq ID No:261 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:259 (IMGT) or Seq ID No:262 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:260 (IMGT) or Seq ID No:263 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:265. 413F09has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:274, comprising the CDRL1 amino acid sequence of Seq ID No:268 (IMGT)or Seq ID No:271 (Kabat), the CDRL2 amino acid sequence of Seq ID No:269(IMGT) or Seq ID No:272 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:270 (IMGT) or Seq ID No:273 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:275. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:266 (heavy chain nucleic acid sequence Seq IDNo:267). A full length light chain amino acid sequence is Seq ID No:276(light chain nucleic acid sequence Seq ID No:277).

414B06 has a heavy chain variable (V_(H)) region amino acid sequence ofSeq ID No:284, comprising the CDRH1 amino acid sequence of Seq ID No:278(IMGT) or Seq ID No:281 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:279 (IMGT) or Seq ID No:282 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:280 (IMGT) or Seq ID No:283 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:285. 414B06has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:294, comprising the CDRL1 amino acid sequence of Seq ID No:288 (IMGT)or Seq ID No:291(Kabat), the CDRL2 amino acid sequence of Seq ID No:289(IMGT) or Seq ID No:292 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:290 (IMGT) or Seq ID No:293 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:295. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:286 (heavy chain nucleic acid sequence Seq IDNo:287). A full length light chain amino acid sequence is Seq ID No:296(light chain nucleic acid sequence Seq ID No:297).

416E01 has a heavy chain variable region (V_(H)) amino acid sequence ofSeq ID No:349, comprising the CDRH1 amino acid sequence of Seq ID No:343(IMGT) or Seq ID No:346 (Kabat), the CDRH2 amino acid sequence of Seq IDNo:344 (IMGT) or Seq ID No:347 (Kabat), and the CDRH3 amino acidsequence of Seq ID No:345 (IMGT) or Seq ID No:348 (Kabat). The heavychain nucleic acid sequence of the V_(H) domain is Seq ID No:350. 416E01has a light chain variable region (V_(L)) amino acid sequence of Seq IDNo:359, comprising the CDRL1 amino acid sequence of Seq ID No:353 (IMGT)or Seq ID No:356 (Kabat), the CDRL2 amino acid sequence of Seq ID No:354(IMGT) or Seq ID No:357 (Kabat), and the CDRL3 amino acid sequence ofSeq ID No:355 (IMGT) or Seq ID No:358 (Kabat). The light chain nucleicacid sequence of the V_(L) domain is Seq ID No:360. The V_(H) domain maybe combined with any of the heavy chain constant region sequencesdescribed herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, SeqID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340,Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq IDNo:532 or Seq ID No:534. The V_(L) domain may be combined with any ofthe light chain constant region sequences described herein, e.g. Seq IDNos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 536 and 538. A full length heavy chain amino acidsequence is Seq ID No:351 (heavy chain nucleic acid sequence Seq IDNo:352). A full length light chain amino acid sequence is Seq ID No:361(light chain nucleic acid sequence Seq ID No:362).

Antibody-Drug Conjugates

Anti-ICOS antibodies can be used as carriers of cytotoxic agents, totarget Tregs. As reported in Example 18, Tregs located in the tumourmicroenvironment (TME) strongly express ICOS. ICOS is more stronglyexpressed on intratumoural Tregs than on intratumoural Teffs orperipheral Tregs. Thus, anti-ICOS antibodies labelled with a toxic drugor pro-drug may preferentially target Tregs in the TME to deliver thetoxic payload, selectively inhibiting those cells. Such targeting ofcytotoxic agents provides an additional route to removing the immunesuppressive effect of Tregs, thereby altering the Treg:Teff balance infavour of Teff activity and may be used as an alternative to, or incombination with, any one or more of the other therapeutic approachesdiscussed herein (e.g., Fc effector-mediated inhibition of Tregs,agonism of effector T cells).

Accordingly, the invention provides an anti-ICOS antibody that isconjugated to a cytotoxic drug or pro-drug. In the case of a pro-drug,the pro-drug is activatable in the TME or other target site oftherapeutic activity to generate the cytotoxic agent. Activation may bein response to a trigger such as photoactivation, e.g., usingnear-infrared light to activate a photoabsorber conjugate [36].Spatially-selective activation of a pro-drug further enhances thecytotoxic effect of the antibody-drug conjugate, combining with the highICOS expression on intratumoural Tregs to provide a cytotoxic effectthat is highly selective for these cells.

For use in an antibody-drug conjugate, the cytotoxic drug or pro-drug ispreferably non-immunogenic and non-toxic (dormant or inactive) duringcirculation of the antibody-drug conjugate in the blood. Preferably thecytotoxic drug (or the pro-drug, when activated) is potent—e.g., two tofour molecules of the drug may be sufficient to kill the target cell. Aphotoactivatable pro-drug is silicapthalocyanine dye (IRDye 700 DX),which induces lethal damage to the cell membrane after near-infraredlight exposure. Cytotoxic drugs include anti-mitotic agents such asmonomethyl auristatin E and microtubule inhibitors such as maytansinederivatives, e.g., mertansine, DM1, emtansine.

Conjugation of the drug (or pro-drug) to the antibody will usually bevia a linker. The linker may be a cleavable linker, e.g., disulphide,hydrazone or peptide link. Cathepsin-cleavable linkers may be used, sothat the drug is released by cathepsin in tumour cells. Alternatively,non-cleavable linkers can be used, e.g., thioether linkage. Additionalattachment groups and/or spacers may also be included.

The antibody in the antibody-drug conjugate may be an antibody fragment,such as Fab′2 or other antigen-binding fragment as described herein, asthe small size of such fragments may assist penetration to the tissuesite (e.g., solid tumour).

An anti-ICOS antibody according to the present invention may be providedas an immunocytokine. Anti-ICOS antibodies may also be administered withimmunocytokines in combination therapy. A number of examples ofantibodies are described herein for use in combination therapy withanti-ICOS, and any of these (e.g., an anti-PD-L1 antibody) may beprovided as immunocytokines for use in the present invention. Animmunocytokine comprises an antibody molecule conjugated to a cytokine,such as IL-2. Anti-ICOS:IL-2 conjugates and anti-PD-L1:IL-2 conjugatesare thus further aspects of the present invention.

An IL-2 cytokine may have activity at the high (αβγ) affinity IL-2receptor and/or the intermediate affinity (αβ) IL-2 receptor. IL-2 asused in an immunocytokine may be human wild type IL-2 or a variant IL-2cytokine having one or more amino acid deletions, substitutions oradditions, e.g., IL-2 having a 1 to 10 amino acid deletion at theN-terminus. Other IL-2 variants include mutations R38A or R38Q.

An example anti-PD-L1 immunocytokine comprises an immunoglobulin heavychain and an immunoglobulin light chain, wherein the heavy chaincomprises in N- to C-terminal direction:

-   -   a) A V_(H) domain comprising CDRH1, CDRH2 and CDRH3; and    -   b) A heavy chain constant region;

and wherein the light chain comprises in N- to C-terminal direction:

-   -   c) A V_(L) domain comprising CDRL1, CDRL2 and CDRL3;    -   d) A light chain constant region, (C_(L));    -   e) Optionally, a linker, (L); and    -   f) An IL-2 cytokine;

wherein the V_(H) domain and V_(L) domain are comprised by anantigen-binding site that specifically binds to human PD-L1; and

wherein the immunocytokine comprises a V_(H) domain which comprises aCDRH3 comprising the motif X₁GSGX₂YGX₃X₄FD (SEQ ID NO: 609), wherein X₁,X₂ and X₃ are independently any amino acid, and X₄ is either present orabsent, and if present, may be any amino acid.

The VH and VL domain may be the VH and VL domain of any anti-PD-L1antibody mentioned herein, e.g., the 1D05 VH and VL domains.

The IL-2 may be human wild type or variant IL-2.

Vaccination

Anti-ICOS antibodies may be provided in vaccine compositions orco-administered with vaccines preparations. ICOS is involved in Tfollicular helper cell formation and the germinal centre reaction [37].Agonist ICOS antibodies thus have potential clinical utility asmolecular adjuvants to enhance vaccine efficacy. The antibodies may beused to increase protective efficacy of numerous vaccines, such as thoseagainst hepatitis B, malaria, HIV.

In the context of vaccination, the anti-ICOS antibody will generally beone that lacks Fc effector function, and thus does not mediate ADCC, CDCor ADCP. The antibody may be provided in a format lacking an Fc region,or having an effector null constant region. Optionally, an anti-ICOSantibody may have a heavy chain constant region that binds one or moretypes of Fc receptor but does not induce ADCC, CDC or ADCP activity, orthat exhibits lower ADCC, CDC and ADCP activity compared with wild typehuman IgG1. Such a constant region may be unable to bind, or may bindwith lower affinity, the particular Fc receptor(s) responsible fortriggering ADCC, CDC or ADCP activity. Alternatively, where cellulareffector functions are acceptable or desirable in the context of thevaccination, the anti-ICOS antibody may comprise a heavy chain constantregion that is Fc effector function positive. Any of IgG1, IgG4 andIgG4.PE formats may for instance be used for anti-ICOS antibodies invaccination regimens, and other examples of suitable isotypes andantibody constant regions are set out in more detail elsewhere herein.

Formulations and Administration

Antibodies may be monoclonal or polyclonal, but are preferably providedas monoclonal antibodies for therapeutic use. They may be provided aspart of a mixture of other antibodies, optionally including antibodiesof different binding specificity.

Antibodies according to the invention, and encoding nucleic acid, willusually be provided in isolated form. Thus, the antibodies, VH and/or VLdomains, and nucleic acids may be provided purified from their naturalenvironment or their production environment. Isolated antibodies andisolated nucleic acid will be free or substantially free of materialwith which they are naturally associated, such as other polypeptides ornucleic acids with which they are found in vivo, or the environment inwhich they are prepared (e.g., cell culture) when such preparation is byrecombinant DNA technology in vitro. Optionally an isolated antibody ornucleic acid (1) is free of at least some other proteins with which itwould normally be found, (2) is essentially free of other proteins fromthe same source, e.g., from the same species, (3) is expressed by a cellfrom a different species, (4) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature, (5) is operably associated (bycovalent or noncovalent interaction) with a polypeptide with which it isnot associated in nature, or (6) does not occur in nature.

Antibodies or nucleic acids may be formulated with diluents or adjuvantsand still for practical purposes be isolated—for example they may bemixed with carriers if used to coat microtitre plates for use inimmunoassays, and may be mixed with pharmaceutically acceptable carriersor diluents when used in therapy. As described elsewhere herein, otheractive ingredients may also be included in therapeutic preparations.Antibodies may be glycosylated, either naturally in vivo or by systemsof heterologous eukaryotic cells such as CHO cells, or they may be (forexample if produced by expression in a prokaryotic cell) unglycosylated.The invention encompasses antibodies having a modified glycosylationpattern. In some applications, modification to remove undesirableglycosylation sites may be useful, or e.g., removal of a fucose moietyto increase ADCC function [38]. In other applications, modification ofgalactosylation can be made in order to modify CDC.

Typically, an isolated product constitutes at least about 5%, at leastabout 10%, at least about 25%, or at least about 50% of a given sample.An antibody may be substantially free from proteins or polypeptides orother contaminants that are found in its natural or productionenvironment that would interfere with its therapeutic, diagnostic,prophylactic, research or other use.

An antibody may have been identified, separated and/or recovered from acomponent of its production environment (eg, naturally orrecombinantly). The isolated antibody may be free of association withall other components from its production environment, eg, so that theantibody has been isolated to an FDA-approvable or approved standard.Contaminant components of its production environment, such as thatresulting from recombinant transfected cells, are materials that wouldtypically interfere with research, diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In some embodiments, the antibody will bepurified: (1) to greater than 95% by weight of antibody as determinedby, for example, the Lowry method, and in some embodiments, to greaterthan 99% by weight; (2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or silverstain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, an isolatedantibody or its encoding nucleic acid will be prepared by at least onepurification step.

The invention provides therapeutic compositions comprising theantibodies described herein. Therapeutic compositions comprising nucleicacid encoding such antibodies are also provided. Encoding nucleic acidsare described in more detail elsewhere herein and include DNA and RNA,e.g., mRNA. In therapeutic methods described herein, use of nucleic acidencoding the antibody, and/or of cells containing such nucleic acid, maybe used as alternatives (or in addition) to compositions comprising theantibody itself. Cells containing nucleic acid encoding the antibody,optionally wherein the nucleic acid is stably integrated into thegenome, thus represent medicaments for therapeutic use in a patient.Nucleic acid encoding the anti-ICOS antibody may be introduced intohuman B lymphocytes, optionally B lymphocytes derived from the intendedpatient and modified ex vivo. Optionally, memory B cells are used.Administration of cells containing the encoding nucleic acid to thepatient provides a reservoir of cells capable of expressing theanti-ICOS antibody, which may provide therapeutic benefit over a longerterm compared with administration of isolated nucleic acid or isolatedantibody.

Compositions may contain suitable carriers, excipients, and other agentsthat are incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311. Compositions may comprise the antibody ornucleic acid in combination with medical injection buffer and/or withadjuvant.

Antibodies, or their encoding nucleic acids, may be formulated for thedesired route of administration to a patient, e.g., in liquid(optionally aqueous solution) for injection. Various delivery systemsare known and can be used to administer the pharmaceutical compositionof the invention. Methods of introduction include, but are not limitedto, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. Formulatingantibodies for subcutaneous administration typically requiresconcentrating them into a smaller volume compared with intravenouspreparations. The high potency of antibodies according to the presentinvention may lend them to use at sufficiently low doses to makesubcutaneous formulation practical, representing an advantage comparedwith less potent anti-ICOS antibodies.

The composition may be administered by any convenient route, for exampleby infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared can be filled in an appropriate ampoule. A pharmaceuticalcomposition of the present invention can be delivered subcutaneously orintravenously with a standard needle and syringe. It is envisaged thattreatment will not be restricted to use in the clinic. Therefore,subcutaneous injection using a needle-free device is also advantageous.With respect to subcutaneous delivery, a pen delivery device readily hasapplications in delivering a pharmaceutical composition of the presentinvention. Such a pen delivery device can be reusable or disposable. Areusable pen delivery device generally utilizes a replaceable cartridgethat contains a pharmaceutical composition. Once all of thepharmaceutical composition within the cartridge has been administeredand the cartridge is empty, the empty cartridge can readily be discardedand replaced with a new cartridge that contains the pharmaceuticalcomposition. The pen delivery device can then be reused. In a disposablepen delivery device, there is no replaceable cartridge. Rather, thedisposable pen delivery device comes prefilled with the pharmaceuticalcomposition held in a reservoir within the device. Once the reservoir isemptied of the pharmaceutical composition, the entire device isdiscarded. Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, the aforesaid antibody may becontained in about 5 to about 100 mg and in about 10 to about 250 mg forthe other dosage forms.

The antibody, nucleic acid, or composition comprising it, may becontained in a medical container such as a phial, syringe, IV containeror an injection device. In an example, the antibody, nucleic acid orcomposition is in vitro, and may be in a sterile container. In anexample, a kit is provided comprising the antibody, packaging andinstructions for use in a therapeutic method as described herein.

One aspect of the invention is a composition comprising an antibody ornucleic acid of the invention and one or more pharmaceuticallyacceptable excipients, examples of which are listed above.“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A pharmaceutically acceptable carrier,excipient, or adjuvant can be administered to a patient, together withan agent, e.g., any antibody or antibody chain described herein, anddoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe agent.

In some embodiments, an anti-ICOS antibody will be the sole activeingredient in a composition according to the present invention. Thus, acomposition may consist of the antibody or it may consist of theantibody with one or more pharmaceutically acceptable excipients.However, compositions according to the present invention optionallyinclude one or more additional active ingredients. Detailed descriptionof agents with which the anti-ICOS antibodies may be combined isprovided elsewhere herein. Optionally, compositions contain multipleantibodies (or encoding nucleic acids) in a combined preparation, e.g.,a single formulation comprising the anti-ICOS antibody and one or moreother antibodies. Other therapeutic agents that it may be desirable toadminister with antibodies or nucleic acids according to the presentinvention include analgaesic agents. Any such agent or combination ofagents may be administered in combination with, or provided incompositions with antibodies or nucleic acids according to the presentinvention, whether as a combined or separate preparation. The antibodyor nucleic acid according to the present invention may be administeredseparately and sequentially, or concurrently and optionally as acombined preparation, with another therapeutic agent or agents such asthose mentioned.

Anti-ICOS antibodies for use in a particular therapeutic indication maybe combined with the accepted standard of care. Thus, for anti-cancertreatment, the antibody therapy may be employed in a treatment regimenthat also includes chemotherapy, surgery and/or radiation therapy forexample. Radiotherapy may be single dose or in fractionated doses,either delivered to affected tissues directly or to the whole body.

Multiple compositions can be administered separately or simultaneously.Separate administration refers to the two compositions beingadministered at different times, e.g. at least 10, 20, 30, or 10-60minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One canalso administer compositions at 24 hours apart, or even longer apart.Alternatively, two or more compositions can be administeredsimultaneously, e.g. less than 10 or less than 5 minutes apart.Compositions administered simultaneously can, in some aspects, beadministered as a mixture, with or without similar or different timerelease mechanism for each of the components.

Antibodies, and their encoding nucleic acids, can be used as therapeuticagents. Patients herein are generally mammals, typically humans. Anantibody or nucleic acid may be administered to a mammal, e.g., by anyroute of administration mentioned herein.

Administration is normally in a “therapeutically effective amount”, thisbeing an amount that produces the desired effect for which it isadministered, sufficient to show benefit to a patient. The exact amountwill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, for example,Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding). Prescription of treatment, e.g. decisions on dosage etc,is within the responsibility of general practitioners and other medicaldoctors and may depend on the severity of the symptoms and/orprogression of a disease being treated. A therapeutically effectiveamount or suitable dose of antibody or nucleic acid can be determined bycomparing its in vitro activity and in vivo activity in an animal model.Methods for extrapolation of effective dosages in mice and other testanimals to humans are known.

As indicated by the in vivo studies described in the Examples herein,anti-ICOS antibody may be effective at a range of doses. Pharmacodynamicstudies are reported in Example 24.

Anti-ICOS antibodies may be administered in an amount in one of thefollowing ranges per dose:

about 10 μg/kg body weight to about 100 mg/kg body weight,

about 50 μg/kg body weight to about 5 mg/kg body weight,

about 100 μg/kg body weight to about 10 mg/kg body weight,

about 100 μg/kg body weight to about 20 mg/kg body weight,

about 0.5 mg/kg body weight to about 20 mg/kg body weight, or

about 5 mg/kg body weight or lower, for example less than 4, less than3, less than 2, or less than 1 mg/kg of the antibody.

An optimal therapeutic dose may be between 0.1 and 0.5 mg/kg in a human,for example about 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg or 0.5 mg/kg. For fixed dosingin adult humans, a suitable dose may be between 8 and 50 mg, or between8 and 25 mg, e.g., 15 mg or 20 mg.

In methods of treatment described herein, one or more doses may beadministered. In some cases, a single dose may be effective to achieve along-term benefit. Thus, the method may comprise administering a singledose of the antibody, its encoding nucleic acid, or the composition.Alternatively, multiple doses may be administered, usually sequentiallyand separated by a period of days, weeks or months. Anti-ICOS antibodymay be repeatedly administered to a patient at intervals of 4 to 6weeks, e.g., every 4 weeks, every 5 weeks, or every 6 weeks. Optionally,the anti-ICOS antibody may be administered to a patient once a month, orless frequently, e.g., every two months or every three months.Accordingly, a method of treating a patient may comprise administering asingle dose of the anti-ICOS antibody to the patient, and not repeatingthe administration for at least one month, at least two months, at leastthree months, and optionally not repeating the administration for atleast 12 months.

As discussed in Example 11c, comparable therapeutic effects may beobtained using either one or multiple doses of anti-ICOS antibody, whichmay be a result of a single dose of antibody being effective to resetthe tumour microenvironment. Physicians can tailor the administrationregimen of the anti-ICOS antibody to the disease and the patientundergoing therapy, taking into account the disease status and any othertherapeutic agents or therapeutic measures (e.g., surgery, radiotherapyetc) with which the anti-ICOS antibody is being combined. In someembodiments, an effective dose of an anti-ICOS antibody is administeredmore frequently than once a month, such as, for example, once everythree weeks, once every two weeks, or once every week. Treatment withanti-ICOS antibody may include multiple doses administered over a periodof at least a month, at least six months, or at least a year.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilised (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment). For treatment to beeffective a complete cure is not contemplated. The method can in certainaspects include cure as well. In the context of the invention, treatmentmay be preventative treatment.

T Cell Therapy

WO2011/097477 described use of anti-ICOS antibodies for generating andexpanding T cells, by contacting a population of T cells with a firstagent that provides a primary activation signal (e.g., an anti-CD3antibody) and a second agent that activates ICOS (e.g., an anti-ICOSantibody), optionally in the presence of a Th17 polarising agent such asIL-1β, IL-6, neutralising anti-IFNγ and/or anti-IL-4. Anti-ICOSantibodies described herein may be used in such methods to provide Tcell populations. Populations of cultured expanded T cells havingtherapeutic activity (e.g., anti-tumour activity) may be generated. Asdescribed in WO2011/097477, such T cells may be used therapeutically inmethods of treating patients by immunotherapy.

Morphological Assay for Anti-ICOS Antibodies as Therapeutic Candidates

It was observed that when candidate therapeutic anti-ICOS antibodieswere coupled to a solid surface and brought into contact withICOS-expressing T cells, they were able to induce morphological changein the cells. On addition of ICOS+ T cells to wells that were internallycoated with anti-ICOS antibodies, cells were seen to change from theirinitial rounded shape, adopting a spindle-shape, spreading and adheringto the antibody-coated surface. This morphological change was notobserved with control antibody. Moreover, the effect was found to bedose-dependent, with faster and/or more pronounced shape changeoccurring as the concentration of antibody on the surface increased. Theshape change provides a surrogate indicator of T cell binding to ICOS,and/or of agonism by anti-ICOS antibody. The assay may be used toidentify an antibody that promotes multimerisation of ICOS on the T cellsurface. Such antibodies represent therapeutic candidate agonistantibodies. Conveniently, the visual indicator provided by this assay isa simple method of screening antibodies or cells, particularly in largenumbers. The assay may be automated to run in a high-throughput system.

Accordingly, one aspect of the invention is an assay for selecting anantibody that binds ICOS, optionally for selecting an ICOS agonistantibody, the assay comprising:

providing an array of antibodies immobilised (attached or adhered) to asubstrate in a test well;

adding ICOS-expressing cells (e.g., activated primary T cells, or MJcells) to the test well;

observing morphology of the cells;

detecting shape change in the cells from rounded to flattened againstthe substrate within the well; wherein the shape change indicates thatthe antibody is an antibody that binds ICOS, optionally an ICOS agonistantibody, and

selecting the antibody from the test well.

The assay may be run with multiple test wells, each containing adifferent antibody for testing, optionally in parallel, e.g., in a 96well plate format. The substrate is preferably an inner surface of thewell. Thus, a two-dimensional surface is provided against whichflattening of the cells may be observed. For example, the bottom and/orwall of a well may be coated with antibody. Tethering of antibody to thesubstrate may be via a constant region of the antibody.

A negative control may be included, such an antibody known not to bindICOS, preferably an antibody that does not bind an antigen on thesurface of the ICOS-expressing cells to be used. The assay may comprisequantifying the degree of morphological change and, where multipleantibodies are tested, selecting an antibody that induces greatermorphological change than one or more other test antibodies.

Selection of antibody may comprise expressing nucleic acid encoding theantibody present in the test well of interest, or expressing an antibodycomprising the CDRs or antigen binding domain of that antibody. Theantibody may optionally be reformatted, for example to provide anantibody comprising the antigen binding domain of the selected antibody,e.g., an antibody fragment, or an antibody comprising a differentconstant region. A selected antibody is preferably provided with a humanIgG1 constant region or other constant region as described herein. Aselected antibody may further be formulated in a composition comprisingone or more additional ingredients—suitable pharmaceutical formationsare discussed elsewhere herein.

CLAUSES

Embodiments of the invention are set out in the following numberedclauses, which are part of the description.

Clause 1. An isolated antibody that binds the extracellular domain ofhuman and/or mouse ICOS, wherein the antibody comprises a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the STIM003 VH domain SEQ ID NO: 408 and a VL domain comprising anamino acid sequence having at least 95% sequence identity to the STIM003VL domain SEQ ID NO: 415.Clause 2. An antibody according to clause 1, wherein the VH domaincomprises a set of heavy chain complementarity determining regions(HCDRs) HCDR1, HCDR2 and HCDR3, wherein

HCDR1 is the STIM003 HCDR1 having amino acid sequence SEQ ID NO: 405,

HCDR2 is the STIM003 HCDR2 having amino acid sequence SEQ ID NO: 406,

HCDR3 is the STIM003 HCDR3 having amino acid sequence SEQ ID NO: 407.

Clause 3. An antibody according to clause 1 or clause 2, wherein the VLdomain comprises a set of light chain complementarity determiningregions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein

LCDR1 is the STIM003 LCDR1 having amino acid sequence SEQ ID NO: 412,

LCDR2 is the STIM003 LCDR2 having amino acid sequence SEQ ID NO: 413,

LCDR3 is the STIM003 LCDR3 having amino acid sequence SEQ ID NO: 414.

Clause 4. An antibody according to clause 1, wherein the VH domain aminoacid sequence is SEQ ID NO: 408 and/or wherein the VL domain amino acidsequence is SEQ ID NO: 415.

Clause 5. An isolated antibody that binds the extracellular domain ofhuman and/or mouse ICOS, comprising

an antibody VH domain comprising complementarity determining regions(CDRs) HCDR1, HCDR2 and HCDR3, and

an antibody VL domain comprising complementarity determining regionsLCDR1, LCDR2 and LCDR3, wherein

HCDR1 is the HCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that HCDR1with 1, 2, 3, 4 or 5 amino acid alterations,

HCDR2 is the HCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that HCDR2with 1, 2, 3, 4 or 5 amino acid alterations, and/or

HCDR3 is the HCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that HCDR3with 1, 2, 3, 4 or 5 amino acid alterations.

Clause 6. An antibody according to clause 5, wherein the antibody heavychain CDRs are those of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009 or comprise the STIM001,STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008or STIM009 heavy chain CDRs with 1, 2, 3, 4 or 5 amino acid alterations.Clause 7. An antibody according to clause 6, wherein the antibody VHdomain has the heavy chain CDRs of STIM003.Clause 8. An isolated antibody that binds the extracellular domain ofhuman and/or mouse ICOS, comprising

an antibody VH domain comprising complementarity determining regionsHCDR1, HCDR2 and HCDR3, and

an antibody VL domain comprising complementarity determining regionsLCDR1, LCDR2 and LCDR3,

wherein LCDR1 is the LCDR1 of STIM001, STIM002, STIM002-B, STIM003,STIM004 STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises thatLCDR1 with 1, 2, 3, 4 or 5 amino acid alterations,

LCDR2 is the LCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that LCDR2with 1, 2, 3, 4 or 5 amino acid alterations, and/or

LCDR3 is the LCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004,STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that LCDR3with 1, 2, 3, 4 or 5 amino acid alterations.

Clause 9. An antibody according to any of clauses 5 to 8, wherein theantibody light chain CDRs are those of STIM001, STIM002, STIM002-B,STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, orcomprise the STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,STIM006, STIM007, STIM008 or STIM009 light chain CDRs with 1, 2, 3, 4 or5 amino acid alterations.Clause 10. An antibody according to clause 9, wherein the antibody VLdomain has the light chain CDRs of STIM003.Clause 11. An antibody according to any of clauses 5 to 10, comprisingVH and/or VL domain framework regions of human germline gene segmentsequences.Clause 12. An antibody according to any of clauses 5 to 11, comprising aVH domain which(i) is derived from recombination of a human heavy chain V gene segment,a human heavy chain D gene segment and a human heavy chain J genesegment, wherein

the V segment is IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01),IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g., V2-5*10);

the D gene segment is IGHD6-19 (e.g., IGHD6-19*01), IGHD3-10 (e.g.,IGHD3-10*01) or IGHD3-9 (e.g., IGHD3-9*01); and/or

the J gene segment is IGHJ6 (e.g., IGHJ6*02), IGHJ4 (e.g., IGHJ4*02) orIGHJ3 (e.g., IGHJ3*02), or

(ii) comprises framework regions FR1, FR2, FR3 and FR4, wherein

FR1 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01),IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations,

FR2 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01),IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations,

FR3 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01),IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations, and/or

FR4 aligns with human germline J gene segment IGJH6 (e.g., JH6*02),IGJH4 (e.g., JH4*02) or IGJH3 (e.g., JH3*02), optionally with 1, 2, 3, 4or 5 amino acid alterations.

Clause 13. An antibody according to any of clauses 5 to 12, comprisingan antibody VL domain which

(i) is derived from recombination of a human light chain V gene segmentand a human light chain J gene segment, wherein

the V segment is IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20 (e.g.,IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g.,IGKV3-11*01), and/or

the J gene segment is IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g., IGKJ2*04),IGLJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g., IGKJ1*01); or

(ii) comprises framework regions FR1, FR2, FR3 and FR4, wherein

FR1 aligns with human germline V gene segment IGKV2-28 (e.g.,IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g.,IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3,4 or 5 amino acid alterations,

FR2 aligns with human germline V gene segment IGKV2-28 (e.g.,IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g.,IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3,4 or 5 amino acid alterations,

FR3 aligns with human germline V gene segment IGKV2-28 (e.g.,IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g.,IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3,4 or 5 amino acid alterations, and/or

FR4 aligns with human germline J gene segment IGKJ4 (e.g., IGKJ4*01),IGKJ2 (e.g., IGKJ2*04), IGKJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g.,IGKJ1*01), optionally with 1, 2, 3, 4 or 5 amino acid alterations.

Clause 14. An antibody according to any of clauses 5 to 13, comprisingan antibody VH domain which is the VH domain of STIM001, STIM002,STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 orSTIM009, or which has an amino acid sequence at least 90% identical tothe antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003,STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.Clause 15. An antibody according to any of clauses 5 to 14, comprisingan antibody VL domain which is the VL domain of STIM001, STIM002,STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 orSTIM009, or which has an amino acid sequence at least 90% identical tothe antibody VL domain sequence of STIM001, STIM002, STIM002-B, STIM003,STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.Clause 16. An antibody according to clause 15, comprising

an antibody VH domain which is selected from the VH domain of STIM001,STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008or STIM009, or which has an amino acid sequence at least 90% identicalto the antibody VH domain sequence of STIM001, STIM002, STIM002-B,STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, and

an antibody VL domain which is the VL domain of said selected antibody,or which has an amino acid sequence at least 90% identical to theantibody VL domain sequence of said selected antibody.

Clause 17. An antibody according to clause 16, comprising the STIM003 VHdomain and the STIM003 VL domain.

Clause 18. An antibody according to any of the preceding clauses,comprising an antibody constant region.

Clause 19. An antibody according to clause 18, wherein the constantregion comprises a human heavy and/or light chain constant region.

Clause 20. An antibody according to clause 18 or clause 19, wherein theconstant region is Fc effector positive.

Clause 21. An antibody according to clause 20, comprising an Fc regionthat has enhanced ADCC, ADCP and/or CDC function compared with a nativehuman Fc region.

Clause 22. An antibody according to any of clauses 18 to 21, wherein theantibody is an IgG1.

Clause 23. An antibody according to clause 21 or clause 22, wherein theantibody is afucosylated.

Clause 24. An antibody according to any of the preceding clauses whichis conjugated to a cytotoxic drug or pro-drug.

Clause 25. An antibody according to any of the preceding clauses, whichis a multispecific antibody.

Clause 26. An isolated antibody that binds the extracellular domain ofhuman and mouse ICOS with an affinity (K_(D)) of less than 50 nM asdetermined by surface plasmon resonance.

Clause 27. An antibody according to clause 26, wherein the antibodybinds the extracellular domain of human and mouse ICOS with an affinity(K_(D)) of less than 5 nM as determined by surface plasmon resonance.

Clause 28. An antibody according to clause 26 or clause 27, wherein theK_(D) of binding the extracellular domain of human ICOS is within10-fold of the K_(D) of binding the extracellular domain of mouse ICOS.

Clause 29. A composition comprising an isolated antibody according toany of the preceding clauses and a pharmaceutically acceptableexcipient.

Clause 30. A composition comprising isolated nucleic acid encoding anantibody according to any of clauses 1 to 28 and a pharmaceuticallyacceptable excipient.

Clause 31. A method of modulating the balance of regulatory T cells(Tregs) to effector T cells (Teffs) to increase Teff response in apatient, comprising administering an antibody according to any ofclauses 1 to 28 or composition according to clause 29 to the patient.Clause 32. A method of treating a disease or condition amenable totherapy by depleting regulatory T cells (Tregs) and/or increasingeffector T cell (Teff) response in a patient, the method comprisingadministering an antibody according to any of clauses 1 to 28 or acomposition according to clause 29 to the patient.Clause 33. An antibody according to any of clauses 1 to 28, or acomposition according to clause 29, for use in a method of treatment ofthe human body by therapy.Clause 34. An antibody or composition for use according to clause 33,for use in modulating the balance of regulatory T cells (Tregs) toeffector T cells (Teffs) to increase effector T cell response in apatient.Clause 35. An antibody or composition for use according to clause 33,for use in treating a disease or condition amenable to therapy bydepleting regulatory T cells (Tregs) and/or increasing effector T cell(Teff) response in a patient.Clause 36. A method according to clause 32, or an antibody or acomposition for use according to clause 35, wherein the disease is acancer or a solid tumour.Clause 37. An antibody according to any of clauses 1 to 28 or acomposition according to clause 29, for use in a method of treatingcancer in a human patient.Clause 38. A method of treating cancer in a human patient, comprisingadministering an antibody according to any of clauses 1 to 28 or acomposition according to clause 29 to the patient.Clause 39. A method or an antibody or composition for use according toany of clauses 36 to 38, wherein the cancer is renal cell cancer, headand neck cancer, melanoma, non small cell lung cancer or diffuse largeB-cell lymphoma.Clause 40. A method or an antibody or composition for use according toany of clauses 31 to 39, wherein the method comprises administering theantibody and another therapeutic agent and/or radiation therapy to thepatient.Clause 41. A method or an antibody or composition for use according toclause 40, wherein the therapeutic agent is an anti-PD-L1 antibody.Clause 42. A method or an antibody or composition for use according toclause 41, wherein the anti-PD-L1 antibody comprises a VH domain havingamino acid sequence SEQ ID NO: 299 and a VL domain having amino acidsequence SEQ ID NO: 300.Clause 43. A method or an antibody or composition for use according toclause 41 or clause 42, wherein the therapeutic agent is an anti-PD-L1IL-2 immunocytokine.Clause 44. A method or an antibody or composition for use according toclause 43, wherein the anti-PD-L1 antibody is an immunocytokinecomprising human wild type or variant IL-2.Clause 45. A method or an antibody or composition for use according toclause 44, wherein the anti-ICOS antibody and the anti-PDL1 antibody areeach able to mediate ADCC, ADCP and/or CDC.Clause 46. A method or an antibody or composition for use according toany of clauses 41 to 45, wherein the anti-ICOS antibody is a human IgG1antibody and the anti-PDL1 antibody is a human IgG1 antibody.Clause 47. A method or an antibody or composition for use according toclause 40, wherein the therapeutic agent is an anti-PD-1 antibody.Clause 48. A method or an antibody or composition for use according toclause 40, wherein the other therapeutic agent is IL-2.Clause 49. A method or an antibody or composition for use according toany of clauses 40 to 48, wherein the method comprises administering theanti-ICOS antibody after administering the other therapeutic agentand/or radiation therapy.Clause 50. A method or an antibody or composition for use according toany of clauses 31 to 49, wherein

the anti-ICOS antibody is conjugated to a pro-drug, and wherein

the method or use comprises

-   -   administering the anti-ICOS antibody to a patient and    -   selectively activating the pro-drug at a target tissue site.        Clause 51. A method or an antibody or composition for use        according to clause 50, wherein the patient has a solid tumour        and the method comprises selectively activating the pro-drug in        the tumour.        Clause 52. A method or an antibody or composition for use        according to clause 50 or clause 51, comprising selectively        activating the pro-drug through photoactivation.        Clause 53. Combination of anti-ICOS human IgG1 antibody and        anti-PDL1 human IgG1 antibody for use in a method of treating        cancer in a patient.        Clause 54. A method of treating cancer in a patient, comprising        administering an anti-ICOS human IgG1 antibody and an anti-PD-L1        human IgG1 antibody to the patient.        Clause 55. Anti-ICOS antibody for use in a method of treating        cancer in a patient, the method comprising administering the        anti-ICOS antibody and the anti-PD-L1 antibody to the patient,        wherein a single dose of the anti-ICOS antibody is administered.        Clause 56. Anti-ICOS antibody for use according to clause 55,        wherein the anti-ICOS antibody is a human IgG1 antibody and the        anti-PD-L1 antibody is a human IgG1 antibody.        Clause 57. Combination according to clause 53, method according        to clause 54 or anti-ICOS antibody for use according to clause        55 or clause 56, wherein the cancer is renal cell cancer, head        and neck cancer, melanoma, non small cell lung cancer or diffuse        large B-cell lymphoma.        Clause 58. A method or an antibody, composition or combination        for use according to any of clauses 41 to 46 or 53 to 54, the        method comprising administering the anti-ICOS antibody and the        anti-PD-L1 antibody to the patient, wherein a single dose of the        anti-ICOS antibody is administered.        Clause 59. A method or an antibody, composition or combination        for use according to clause 58, wherein the method comprises        administering a single dose of the anti-ICOS antibody followed        by multiple doses of the anti-PD-L1 antibody.        Clause 60. A method or an antibody, composition or combination        for use according to any of clauses 41 to 46 or 53 to 54,        wherein the anti-ICOS antibody and the anti-PDL1 antibody are        provided in separate compositions for administration.        Clause 61. A method or an antibody, composition or combination        for use according to any of clauses 41 to 46 or 53 to 60,        wherein the anti-ICOS antibody and/or the anti-PD-L1 antibody        comprises a human IgG1 constant region comprising amino acid        sequence SEQ ID NO: 340.        Clause 62. Anti-ICOS antibody for use in a method of treating a        patient, the method comprising administering the anti-ICOS        antibody to a patient who has an increased level of        ICOS-positive regulatory T cells following treatment with        another therapeutic agent.        Clause 63. A method of treating a patient, the method comprising        administering an anti-ICOS antibody to a patient who has an        increased level of ICOS-positive regulatory T cells following        treatment with another therapeutic agent.        Clause 64. An anti-ICOS antibody for use according to clause 62,        or a method according to clause 63, wherein the method comprises        administering a therapeutic agent to the patient, determining        that the patient has an increased level of ICOS-positive        regulatory T cells following the treatment with said agent, and        administering an anti-ICOS antibody to the patient to reduce the        level of regulatory T cells.        Clause 65. An anti-ICOS antibody for use or a method according        to any of clauses 62 to 64, wherein the therapeutic agent is        IL-2 or an immunomodulatory antibody (e.g., anti-PDL-1,        anti-PD-1 or anti-CTLA-4).        Clause 66. An anti-ICOS antibody for use or a method according        to any of clauses 62 to 65, wherein the method comprises        treating a tumour, e.g., melanoma, such as metastatic melanoma.        Clause 67. Anti-ICOS antibody for use in a method of treating        cancer in a patient by in vivo vaccination of the patient        against their cancer cells, the method comprising

treating the patient with a therapy that causes immunological cell deathof the cancer cells, resulting in presentation of antigen toantigen-specific effector T cells, and

administering an anti-ICOS antibody to the patient, wherein theanti-ICOS antibody enhances the antigen-specific effector T cellresponse.

Clause 68. A method of treating cancer in a patient by in vivovaccination of the patient against their cancer cells, the methodcomprising

treating the patient with a therapy that causes immunological cell deathof the cancer cells, resulting in presentation of antigen toantigen-specific effector T cells, and

administering an anti-ICOS antibody to the patient, wherein theanti-ICOS antibody enhances the antigen-specific effector T cellresponse.

Clause 69. A method of treating cancer in a patient by in vivovaccination of the patient against their cancer cells, the methodcomprising administering an anti-ICOS antibody to the patient, wherein

the patient is one who has been previously treated with a therapy thatcauses immunological cell death of the cancer cells, resulting inpresentation of antigen to antigen-specific effector T cells, andwherein

the anti-ICOS antibody enhances the antigen-specific effector T cellresponse.

Clause 70. Anti-ICOS antibody for use or a method according to any ofclauses 67 to 69, wherein the therapy that causes immunological celldeath is radiation of the cancer cells, administration of achemotherapeutic agent and/or administration of an antibody directed toa tumour-associated antigen.Clause 71. Anti-ICOS antibody for use or a method according to clause70, wherein the chemotherapeutic agent is oxaliplatin.Clause 72. Anti-ICOS antibody for use or a method according to clause70, wherein the tumour-associated antigen is HER2 or CD20.Clause 73. Anti-ICOS antibody for use in a method of treating a cancerin a patient, wherein the cancer is or has been characterised as beingpositive for expression of ICOS ligand and/or FOXP3.Clause 74. A method of treating a cancer in a patient, wherein thecancer is or has been characterised as being positive for expression ofICOS ligand and/or FOXP3, the method comprising administering ananti-ICOS antibody to the patient.Clause 75. Anti-ICOS antibody for use according to clause 73, or amethod according to clause 74, wherein the method comprises:

testing a sample from a patient to determine that the cancer expressesICOS ligand and/or FOXP3;

selecting the patient for treatment with the anti-ICOS antibody; and

administering the anti-ICOS antibody to the patient.

Clause 76. Anti-ICOS antibody for use according to clause 73, or amethod according to clause 74, wherein the method comprisesadministering an anti-ICOS antibody to a patient from whom a test samplehas indicated that the cancer is positive for expression of ICOS ligandand/or FOXP3.Clause 77. Anti-ICOS antibody for use or a method according to clause 75or clause 76, wherein the sample is biopsy sample of a solid tumour.Clause 78. Anti-ICOS antibody for use in a method of treating a cancerin a patient, wherein the cancer is or has been characterised as beingrefractory to treatment with an immunooncology drug, e.g., anti-CTLA-4antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody oranti-GITR antibody.Clause 79. A method of treating a cancer in a patient, wherein thecancer is or has been characterised as being refractory to treatmentwith an immunooncology drug, e.g., anti-CTLA-4 antibody, anti-PD1antibody, anti-PD-L1 antibody, anti-CD137 antibody or anti-GITRantibody, the method comprising administering an anti-ICOS antibody tothe patient.Clause 80. Anti-ICOS antibody for use according to clause 78 or a methodaccording to clause 79, wherein the method comprises:

treating the patient with the immunooncology drug;

determining that the cancer is not responsive to the drug;

selecting the patient for treatment with the anti-ICOS antibody; and

administering the anti-ICOS antibody to the patient.

Clause 81. Anti-ICOS antibody for use according to clause 78, or amethod according to clause 79, wherein the method comprisesadministering an anti-ICOS antibody to a patient whose cancer was notresponsive to prior treatment with the immunooncology drug.

Clause 82. Anti-ICOS antibody for use or a method according to any ofclauses 73 to 81, wherein the cancer is a tumour derived from cells thathave acquired ability to express ICOS ligand.

Clause 83. Anti-ICOS antibody for use or a method according to clause82, wherein the cancer is melanoma.

Clause 84. Anti-ICOS antibody for use or a method according to any ofclauses 73 to 81, wherein the cancer is derived from anantigen-presenting cell, such as a B lymphocyte (e.g., B cell lymphoma,such as diffuse large B cell lymphoma) or a T lymphocyte.

Clause 85. Anti-ICOS antibody for use or a method according to any ofclauses 73 to 81, wherein the cancer is resistant to treatment with ananti-CD20 antibody.

Clause 86. Anti-ICOS antibody for use or a method according to clause85, wherein the cancer is B cell lymphoma.

Clause 87. Anti-ICOS antibody for use or a method according to clause86, wherein the anti-CD20 antibody is rituximab.

Clause 88. Anti-ICOS antibody for use or a method according to any ofclauses 85 to 87, wherein the method comprises treating the patient withthe anti-CD20 antibody;

determining that the cancer is not responsive to the anti-CD20 antibody;

testing a sample from a patient to determine that the cancer expressesICOS ligand;

selecting the patient for treatment with the anti-ICOS antibody; and

administering the anti-ICOS antibody to the patient.

Clause 89. Anti-ICOS antibody for use or a method according to any ofclauses 85 to 87, wherein the method comprises administering ananti-ICOS antibody to a patient whose cancer was not responsive to priortreatment with anti-CD20 antibody.

Clause 90. Anti-ICOS antibody for use or a method according to any ofclauses 67 to 89, wherein the cancer is a solid tumour.

Clause 91. Anti-ICOS antibody for use or a method according to any ofclauses 67 to 89, wherein the cancer is a haemotological liquid tumour.

Clause 92. Anti-ICOS antibody for use or a method according to clause 90or 91, wherein the tumour is high in regulatory T cells.

Clause 93. Anti-ICOS antibody for use or a method according to any ofclauses 53 to 92, wherein the anti-ICOS antibody is as defined in any ofclauses 1 to 28 or is provided in a composition according to clause 29.

Clause 94. A transgenic non-human mammal having a genome comprising ahuman or humanised immunoglobulin locus encoding human variable regiongene segments, wherein the mammal does not express ICOS.

Clause 95. A method of producing an antibody that binds theextracellular domain of human and non-human ICOS, comprising

(a) immunising a mammal according to clause 94 with human ICOS antigen;

(b) isolating antibodies generated by the mammal;

(c) testing the antibodies for ability to bind human ICOS and non-humanICOS; and

(d) selecting one or more antibodies that binds both human and non-humanICOS.

Clause 96. A method according to clause 95, comprising immunising themammal with cells expressing human ICOS.

Clause 97. A method according to clause 95 or clause 96, comprising

(c) testing the antibodies for ability to bind human ICOS and non-humanICOS using surface plasmon resonance and determining binding affinities;and

(d) selecting one or more antibodies for which the K_(D) of binding tohuman ICOS is less than 50 nM and the K_(D) of binding to non-human ICOSis less than 500 nM.

Clause 98. A method according to clause 97, comprising

(d) selecting one or more antibodies for which the K_(D) of binding tohuman ICOS is less than 10 nM and the K_(D) of binding to non-human ICOSis less than 100 nM.

Clause 99. A method according to any of clauses 95 to 98, comprising

(c) testing the antibodies for ability to bind human ICOS and non-humanICOS using surface plasmon resonance and determining binding affinities;and

(d) selecting one or more antibodies for which the K_(D) of binding tohuman ICOS is within 10-fold of the K_(D) of binding to non-human ICOS.

Clause 100. A method according to clause 99, comprising

(d) selecting one or more antibodies for which the K_(D) of binding tohuman ICOS is within 5-fold of the K_(D) of binding to non-human ICOS.

Clause 101. A method according to any of clauses 95 to 100, comprisingtesting the antibodies for ability to bind non-human ICOS from the samespecies as the mammal.

Clause 102. A method according to any of clauses 95 to 101, comprisingtesting the antibodies for ability to bind non-human ICOS from adifferent species as the mammal.

Clause 103. A method according to any of clauses 95 to 102, wherein themammal is a mouse or a rat.

Clause 104. A method according to any of clauses 95 to 103, wherein thenon-human ICOS is mouse ICOS or rat ICOS.

Clause 105. A method according to any of clauses 95 to 104, wherein thehuman or humanised immunoglobulin locus comprises human variable regiongene segments upstream of an endogenous constant region.

Clause 106. A method according to clause 105, comprising

(a) immunising a mammal according to clause 94 with human ICOS antigen,wherein the mammal is a mouse;

(b) isolating antibodies generated by the mouse;

(c) testing the antibodies for ability to bind human ICOS and mouseICOS; and

(d) selecting one or more antibodies that binds both human and mouseICOS.

Clause 107. A method according to any of clauses 95 to 106, comprisingisolating nucleic acid encoding an antibody heavy chain variable domainand/or an antibody light chain variable domain.

Clause 108. A method according to any of clauses 95 to 107, wherein themammal generates antibodies through recombination of human variableregion gene segments and an endogenous constant region.

Clause 109. A method according to clause 107 or clause 108, comprisingconjugating the nucleic acid encoding the heavy and/or light chainvariable domain to a nucleotide sequence encoding a human heavy chainconstant region and/or human light chain constant region respectively.Clause 110. A method according to any of clauses 107 to 109, comprisingintroducing the nucleic acid into a host cell.Clause 111. A method according to clause 110, comprising culturing thehost cell under conditions for expression of the antibody, or of theantibody heavy and/or light chain variable domain.Clause 112. An antibody, or antibody heavy and/or light chain variabledomain, produced by the method according to any of clauses 95 to 111.Clause 113. A method of selecting an antibody that binds ICOS,optionally for selecting an ICOS agonist antibody, the assay comprising:

providing an array of antibodies immobilised (attached or adhered) to asubstrate in a test well;

adding ICOS-expressing cells (e.g., activated primary T cells, or MJcells) to the test well;

observing morphology of the cells;

detecting shape change in the cells from rounded to flattened againstthe substrate within the well; wherein the shape change indicates thatthe antibody is an antibody that binds ICOS, optionally an ICOS agonistantibody;

selecting the antibody from the test well;

expressing nucleic acid encoding the CDRs of the selected antibody; and

formulating the antibody into a composition comprising one or moreadditional components.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.All documents mentioned in this specification, including published UScounterparts of any patents or patent applications referred to, areincorporated herein by reference in their entirety.

EXPERIMENTAL EXAMPLES

The following Examples describe the generation, characterisation andperformance of anti-ICOS antibodies. Antibodies were generated using theKymouse™, a transgenic mouse platform capable of generating antibodieswith human variable domains. Antibodies from the Kymouse™ have humanvariable domains, generated from human V (D) and J segments, and mouseconstant domains. The endogenous mouse variable genes have been silencedand make up a very small portion of the repertoire (less than 0.5% ofall heavy chain variable regions are of mouse origin). The Kymouse™system is described in Lee et al 2014 [39], WO2011/004192, WO2011/158009and WO2013/061098. This project employed the Kymouse™ HK strain, inwhich the heavy chain locus and light chain kappa locus are humanised.

ICOS knock-out Kymouse™ were immunised with either ICOS protein or acombination of alternating boosts of protein and cells expressing humanand mouse ICOS.

Hits which bound to human ICOS were identified. The primary selectioncriteria for the screen was binding to human cell expressed ICOS (CHOcells) and binding to ICOS protein (HTRF). Binding to mouse ICOS proteinand mouse cell expressed ICOS (CHO cells) was also assessed and takeninto consideration when selecting primary screen hits. Using thesecriteria hits were progressed to a secondary screen. In the secondaryscreen hits were confirmed by determining binding to human and mouseICOS expressed on CHO cells by flow cytometry.

From a large number of antibodies screened, a small panel wereidentified which bind to human/cynomolgus and mouse ICOS as determinedby surface plasmon resonance and flow cytometry. These antibodiesincluded STIM001, STIM002 and its variant STIM002-B, STIM003, STIM004and STIM005. An additional four antibodies STIM006, STIM007, STIM008 andSTIM009 were also selected, showing less cross-reactivity with mouseICOS but demonstrating agonism of the human ICOS receptor. The datapresented here indicate the ability of anti-ICOS antibodies to act asagonists of the ICOS receptor in an ICOS positive CD4+ cell line andalso in a primary T cell-based assay, show cell-killing ability in anADCC assay and an ability to promote an anti-tumour immune response invivo.

Example 1: Generation of ICOS Knock-Out Mouse

An ICOS knock-out Kymouse™ line was generated by homologousrecombination in Kymouse™ HK ES cells. In brief, a 3.5 kb targetingvector encoding a puromycin selection was targeted into ES cells.Successful targeting resulted in the replacement of a small region (72bp) of the mouse ICOS locus with the puromycin cassette, disrupting thesignal peptide/start codon of the gene. Positive ES clones were expandedand microinjected into mouse blastocysts and resulting chimaeras bred inorder to ultimately generate animals homozygous for both the humanisedheavy and kappa immunoglobulin loci and the modified, functionally-null,ICOS locus.

Example 2: Antigen and Cell-Line Preparation

Generation of Stably Transfected MEF and CHO-S Cells Expressing Human orMouse ICOS

Full length DNA sequences encoding human and mouse ICOS were codonoptimised for mammalian expression, ordered as synthetic string DNA andcloned into an expression vector under the control of the CMV promoterand flanked by 3′ and 5′ piggyBac specific terminal repeat sequencesfacilitating stable integration into the cell genome (see [40]). Theexpression vector contained a puromycin selection cassette to facilitatestable cell line generation. For generation of human ICOS expressing andmouse ICOS expressing cell lines respectively, the human or mouse ICOSexpression plasmid was co-transfected with a plasmid encoding piggyBactransposase into a mouse embryonic fibroblast (MEF) cell line and CHO-Scells using the FreeStyle Max transfection reagent (Invitrogen)according to manufacturer instructions. MEF cells were generated fromembryos obtained from a 129S5 crossed to C57BL6 female mouse. Twentyfour hours after transfection, the media was supplemented with puromycinand grown for at least two weeks to select stable cell lines. Cellculture medium was replaced every 3-4 days. Expression of human or mouseICOS protein was assessed by flow cytometry using anti-human oranti-mouse ICOS-PE conjugated antibodies (eBioscience) respectively.Complete MEF media was made up of Dulbecco's Modified Eagle's Medium(Gibco) supplemented with 10% v/v fetal bovine serum (Gibco). CompleteCHO-S media was made up of CD-CHO media supplemented with 8 mM Glutamax(Gibco). CHO-S cells are the CHO-3E7 cell line included with the pTT5system available from the National Research Council of Canada, but otherCHO cell lines could be employed.

Preparation of MEF Cells for Mouse Immunisations

Cell culture medium was removed and cells washed once with 1×PBS. Cellswere treated for 5 minutes with trypsin to loosen cells from tissueculture surface. Cells were collected and the trypsin neutralized by theaddition of complete media containing 10% v/v fetal bovine serum (FCS).Cells were then centrifuged at 300 g for 10 minutes and washed with 25ml of 1×PBS. Cells were counted and resuspended at the appropriateconcentration in 1×PBS.

Cloning and Expression of Recombinant Proteins

Synthetic DNA encoding the extracellular domains of human ICOS (NCBI ID:NP_036224.1), mouse ICOS (NCBI ID: NP_059508.2) and cynomolgus ICOS(GenBank ID: EHH55098.1) were cloned into either a pREP4 (Invitrogen) ora pTT5 (National Research Council Of Canada) expression plasmid usingstandard molecular biology techniques. The constructs also containedeither a human Fc, a mouse Fc or a FLAG His peptide motif to aidpurification and detection. These were added to the DNA constructs byoverlap extension. All constructs were sequenced prior to expression toensure their correct sequence composition.

Example 3: Immunisation

ICOS knock out HK Kymice™ (see Example 1), Kymouse™ wild type HK strainand Kymouse™ wild type HL strain were immunised according to theregimens shown in Table E3. Kymouse™ wild type HK and HL strains expresswild type mouse ICOS. In the HK strain the immunoglobulin heavy chainlocus and light chain kappa locus are humanised, and in the HL strainthe immunoglobulin heavy chain locus and light chain lambda locus arehumanised.

TABLE E3 Immunisation regimens for Kymouse ™ strains Regime Mouse PrimeBoost 1 Boost 2 Boost 3 Final Boost KM103 ICOS KO mICOS Fc hICOS MEFmICOS Fc hICOS MEF mICOS Fc KM103 ICOS KO mICOS Fc hICOS Fc mICOS FchICOS Fc N/A KM111 ICOS KO mICOS Fc + hICOS mICOS mICOS Fc + mICOS mICOSFc + Fc MEF + hICOS Fc MEF + hICOS Fc hICOS MEF hICOS MEF KM111 ICOS KOhICOS Fc hICOS MEF hICOS Fc hICOS MEF hICOS Fc KM111 ICOS KO mICOS FcmICOS mICOS Fc mICOS mICOS Fc MEF MEF KM111 HK and hICOS Fc hICOS MEFhICOS Fc hICOS MEF hICOS Fc HL KM135 ICOS KO mICOS Fc 1 prime and 6boosts (RIMMS) KM135 ICOS KO hICOS Fc 1 prime and 6 boosts (RIMMS)Key to table:mICOS Fc=mouse ICOS protein with human FchICOS Fc=human ICOS protein with human FcmICOS MEF=mouse ICOS expressed on MEF cellshICOS MEF=human ICOS expressed on MEF cellsmICOS Fc+hICOS Fc=mouse ICOS protein with human Fc+human ICOS proteinwith human Fc administered simultaneouslymICOS MEF+hICOS MEF=mouse ICOS expressed on MEF cells+human ICOSexpressed on MEF cells administered simultaneouslyICOS KO=ICOS knockout HK KymouseHK and HL=wild type Kymouse HK and HL genotypeRIMMS is a modified sub-cutaneous immunisation procedure (rapidimmunisation at multiple sites); modified after Kilpatrick et al. [41]).Immunisation regimens KM103 and KM111 were prime-rest-boost byintraperitoneal (i.p.) administration. Sigma Adjuvant System was usedfor all immunisations and rest intervals were usually between 2 and 3weeks. Final boosts were administered by intravenously in absence ofadjuvant.

Sera from serial or terminal blood samples were analysed for thepresence of specific antibodies by flow cytometry and the titre data wasused (where possible) to select mice to be used for B cell sorting.

Example 4: Comparison of Serum Titres Between ICOS KO and Wild Type Mice

Serum titres of immunised ICOS KO and immunised wild type Kymouse weredetermined using flow cytometry. In ICOS KO mice, immunisation withhuman ICOS antigen induced a serum immunoglobulin response with Igbinding to both human and mouse ICOS expressed on CHO cells (FIG. 1a ).Conversely, in the wild type Kymouse (expressing mouse ICOS),immunisation with the same human ICOS antigen produced sera that showedmarkedly reduced binding to mouse ICOS compared with binding of the sameserum to human ICOS (FIG. 1b ).

Method

CHO-S cells expressing human ICOS or mouse ICOS (see Example 2) oruntransfected CHO-S cells (referred to as wild type (WT)), suspended inFACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) were distributed to a96-well, V-bottom plate (Greiner) at a density of 10⁵ cells per well. Atitration of mouse serum was prepared, diluting samples in FACS buffer.50 μL/well of this titration was then added to the cell plate. Todetermine the change in activity level due to immunisation, serum fromeach animal prior to immunisation was diluted to 1/100 in FACS bufferand 50 μL/well added to the cells. Cells were incubated at 4° C. for 1hour. Cells were washed twice with 150 μL PBS, centrifuging after eachwash step and aspirating supernatant (centrifuged at 300×g for 3minutes). To detect antibody binding, APC goat-anti-mouse IgG (JacksonImmunoResearch) was diluted 1/500 in FACS buffer and 50 μL was added tothe cells. In some instances AF647 goat-anti-mouse IgG (JacksonImmunoResearch) was used. Cells were incubated 1 hour at 4° C. in thedark, then washed twice with 150 μL PBS as above. To fix cells, 100 μL2% v/v paraformaldehyde was added and cells incubated for 30 minutes at4° C. Cells were then pelleted by centrifugation at 300×g and the platesresuspended in 50 μL of FACS buffer. Fluorescent signal intensity(geometric mean) was measured by flow cytometry using a BD FACS Arrayinstrument.

Example 5: Sorting of Antigen-Specific B Cells by FACS

B-cells expressing anti-ICOS antibodies were recovered from immunisedmice, using techniques substantially as described in Example 1 ofWO2015/040401. In brief, splenocytes and/or lymph node cells isolatedfrom the immunisation regimes were stained with an antibody cocktailcontaining markers for the selection of cells of interest (CD19),whereas unwanted cells were excluded from the final sorted population(IgM, IgD, 7AAD). CD19⁺ B-cells were further labelled withfluorescently-tagged human ICOS ECD-Fc dimers and fluorescently-taggedmouse ICOS ECD-Fc to detect B-cells producing anti-ICOS antibodies.Fluorescent labelling of human and mouse ICOS was with AlexaFluor647 andAlexaFluor488, respectively—see Example 6. Cells binding human ICOS, orboth human and mouse ICOS were selected. These cells were single cellsorted by FACS into lysis buffer. V-region sequences were recoveredusing RT-PCR and two further rounds of PCR, then bridged to mouse IgG1constant region and expressed in HEK293 cells. Supernatants from HEK293cells were screened for the presence of ICOS binding and functionalantibodies. This method is hereafter referred to as BCT.

Example 6: Screening of Antibodies from BCT

HTRF Screening of BCT Supernatants for Binding to Recombinant Human andMouse ICOS-Fc

Supernatants collected from BCT in Example 5 were screened for theability of secreted antibodies to bind to human ICOS Fc and mouse ICOSFc expressed as recombinant proteins. Binding of secreted antibodies torecombinant human and mouse ICOS were identified by HTRF® (HomogeneousTime-Resolved Fluorescence, Cisbio) assay format using FluoProbes®647H(Innova Biosciences) labelled ICOS (referred to herein as 647 hICOS or647 mICOS for human ICOS and mouse ICOS labelled with FluoProbes®647Hrespectively). 5 μL BCT supernatant was transferred to a white 384-well,low-volume, non-binding surface polystyrene plate (Greiner). 5 μL of 20nM 647 hICOS or 647 mICOS diluted in HTRF assay buffer was added to allwells. For human ICOS binding assay the reference antibody was dilutedin BCT media (Gibco #A14351-01) to 120 nM and 5 μL added to plate. Fornegative control wells for human ICOS binding assay, 5 μL of mouse IgG1(Sigma M9269 in some instances referred to as CM7) diluted to 120 nM inBCT media. In the case of mouse ICOS binding assay the referenceantibody was diluted in BCT media (Gibco #A14351-01) to 120 nM and 5 μLadded to plate. A rat IgG2b isotype control (R&D systems) was added tonegative control wells (R&D Systems) diluted in BCT media to 120 nM and5 μL added to plate. Binding of secreted antibodies to human ICOS wasdetected by addition of 10 μL of goat anti-mouse IgG (Southern Biotech)directly labelled with Europium cryptate (Cisbio) diluted 1/2000 in HTRFassay buffer. In the case of the mouse ICOS binding assay 5 μL of mouseanti-Rat IgG2B-UBLB (Southern Biotech) was added to positive andnegative control wells, and 5 μL of HTRF assay buffer added to all otherwells of plate. Then 5 μL of goat anti-mouse IgG (Southern Biotech)directly labelled with Europium cryptate (Cisbio) diluted 1/1000 in HTRFassay buffer was added to detect binding. The plate was left to incubatein the dark for 2 hours prior to reading time resolved fluorescence at620 nm and 665 nm emission wavelengths, 100 flashes, using an EnVisionplate reader (Perkin Elmer).

Data were analysed by calculating 665/620 ratio and percent effect foreach sample according to equation 2 and equation 1 respectively.

For KM103 and KM11-B1, primary hits were selected based on greater thanor equal to 5 percent effect for binding to human and mouse ICOS. ForKM135 primary hits were selected based on greater than or equal to 10percent effect for binding to human and mouse ICOS. For KM111-B2 primaryhits were defined as greater than or equal to 4 percent effect forbinding to human and greater than or equal to 3 percent effect forbinding to mouse ICOS.

Equation 1: Calculation of Percentage Effect from Primary ScreenEnvision Cell Binding and HTRF

Using well ratio value (equation 3) or 665/620 nm ratio (see equation 2)(HTRF)

${{Percent}\mspace{14mu}{effect}} = \frac{\left( {{{sample}\mspace{14mu}{well}} - {{non}\text{-}{specific}\mspace{14mu}{binding}}} \right) \times 100}{\left( {{{total}\mspace{14mu}{binding}} - {{non}\text{-}{specific}\mspace{14mu}{binding}}} \right)}$Non-specific  binding = values  from  wells  containing  isotypecontrol  mouse  IgG 1Total  Binding = values  from  wells  containing  reference  antibodyEquation 2: Calculation of 665/620 Ratio665/620ratio=(sample 665/620 nm value)×10,000Equation 3: Calculation of 647/FITC RatioData were first normalised for cell number by dividing mAb channel (647)by FITC (cell stain) channel to give “well ratio value”:

${{Well}\mspace{14mu}{Ratio}\mspace{14mu}{Value}} = \frac{647\mspace{14mu}{Channel}}{{FITC}\mspace{14mu}{Channel}}$Screening of BCT Supernatants for Binding to Cell-Expressed Human andMouse ICOS

Supernatants collected from BCT in Example 5 were screened for theability of secreted antibodies to bind to human or mouse ICOS expressedon the surface of CHO-S cells. To determine CHO-S human and mouse ICOSbinding, cells were plated in black-walled, clear-bottom tissue culturetreated 384-well plates (Perkin Elmer) at 4×10⁴/well in F12 media(Gibco) supplemented with 10% FBS (Gibco) and cultured overnight.Culture media was removed from 384-well assay plates. At least 50 μL ofBCT supernatant or 50 μL reference antibody at 2 μg/mL in BCT media orisotype IgG1 control antibody (referred to in some instances as Cm7,Sigma M9269), at a final concentration of 2 μg/mL) diluted in BCT mediawere added to each well. Plates were incubated for 1 hour at 4° C.Supernatant was aspirated and 50 μL of goat anti-Mouse 647 (Jacksonimmunoresearch) at 5 μg/ml with vibrant green DNA stain (LifeTechnologies) diluted 1 in 500 in secondary antibody buffer (1×PBS+1%BSA+0.1% Sodium Azide) was added to detect antibody binding andvisualise cells. Plates were incubated for 1 hr at 4 degrees.Supernatant was aspirated and 25 μL of 4% v/v paraformaldehyde added andplates were incubated for 15 minutes at room temperature. Plates werewashed twice with 100 μL PBS and then the wash buffer was completelyremoved. Fluorescence intensity was measured using Envision plate reader(Perkin Elmer) measuring FITC (excitation 494 nm, emission 520 nm) andalexafluor 647 (excitation 650 nm, emission 668 nm). Assay signal wasdetermined as described in equation 3 and percent effect as inequation 1. Total binding was defined using reference antibody at afinal assay concentration of 2 μg/mL. Non-specific binding was definedusing mouse IgG1 isotype control (Sigma) at a final assay concentrationof 2 μg/mL. Criteria for hit selection were based on assay signal andpercent effect.

For KM103, KM111-B1 and KM135, primary hits were selected based ongreater than or equal to 10 percent effect. For KM111-132, primary hitswere selected based on greater than or equal to 4 percent effect.

Summary of Primary Screening Results

TABLE E6 Summary of number of BCT supernatants screened fromimmunisations, and number of supernatants meeting primary screeningselection criteria for binding to human and mouse ICOS. SupernatantsExperiment ID screened Primary hits selected KM103 1232 40 KM111-B1 1056198 KM111-B2 1056 136 KM135 704 31FACS Screening for Binding to Cell Expressed Human and Mouse ICOS

BCT supernatants and HEK293 expressed antibodies from Example 5 weretested for ability to bind to CHO-S cells expressing human or mouseICOS.

CHO-S cells expressing human or mouse ICOS (see Example 2), were dilutedin FACS buffer (PBS 1% BSA 0.1% sodium azide) and were distributed to a96-well, V-bottom plate (Greiner) at a density of 1×10⁵ cells per well.Cells were washed with 150 μL PBS and centrifuged at 300 g for 3minutes. For supernatant screening, supernatant was aspirated and 150 μLPBS added. This wash step was repeated. 30 μL BCT undiluted supernatantor 50 μL of reference antibody or control antibody diluted to 5 μg/ml inBCT media was added to the washed cells. Cells were incubated at 4° C.for 60 minutes. 150 μL FACS buffer was added and cells washed asdescribed above. To detect antibody binding, 50 μL of goat anti-mouseAPC (Jackson ImmunoResearch) diluted to 2 μg/ml in FACS buffer was addedto cells. Cells were incubated 4° C. for 60 minutes. Cells were washedtwice with 150 μL FACS buffer, centrifuging at 300 g for 3 minutes aftereach wash step and aspirating supernatant. Cells were fixed by additionof 25 μL 4% paraformaldehyde for 20 minutes at room temperature. Cellswere washed once as above and resuspended in FACS buffer for analysis.APC signal intensity (geometric mean) was measured by flow cytometryusing a BD FACS Array instrument. Data were plotted as geometric meanvalues without further calculation.

A small sub-set of antibodies were selected as meeting more stringentspecies cross-reactivity criteria in this further screening comparedwith the primary screening. In brief:

From KM103, 4 antibodies were selected by taking the average geomean ofthe hybrid control binding to hICOS, mICOS and WT CHO cells andidentifying mouse and human binders that were >4 fold above. These 4antibodies were designated STIM001, STIM002-B, STIM007 and STIM009.

From KM111-B1, 4 antibodies were selected by taking the average ofgeomean of the negative control (Armenian hamster: clone HTK888) bindingto hICOS, mICOS and WT CHO cells and identifying mouse and human bindersthat were >10 fold above.

From KM111-132, 4 antibodies were selected by taking the average ofgeomean of the negative control (Armenian hamster: clone HTK888) bindingto hICOS, mICOS and WT CHO cells and identifying mouse and human bindersthat were >4 fold above. These 4 antibodies included STIM003, STIM004and STIM005.

From KM135, no cross-reactive antibodies were identified. Due to atechnical failure of the FACS secondary screening method, screening wasalso carried out using SPR and HTRF, but no antibodies were found tomeet the desired cross-reactivity level.

In conclusion, from the various multiple immunisation regimens describedin Example 3, upward of 4000 BCT supernatants (from the ICOS KO miceonly) were screened for binding to human ICOS and mouse ICOS, and asmall panel of candidates, including STIM001, STIM002-B, STIM003,STIM004, STIM005, STIM007 and STIM009, were identified as having themost promising characteristics for further development. These were takenforward for more detailed characterisation.

Separately, two antibodies STIM006 and STIM008, which did not meet thespecies cross-reactivity criteria, were also chosen for furthercharacterisation on the basis of their ability to bind human ICOS.

Example 7: Affinity Determination by Surface Plasmon Resonance (SPR)

Fab affinities of the ICOS leads were generated by SPR using the ProteOnXPR3 6 (BioRad). An anti-human IgG capture surface was created on a GLCbiosensor chip by primary amine coupling, immobilising three anti-humanIgG antibodies (Jackson Labs 109-005-008, 109-006-008 and 309-006-008).The human Fc tagged human ICOS (hICOS) and mouse ICOS (mICOS) werecaptured individually on the anti-human IgG surface and the purifiedFabs were used as analytes at 5000 nM, 1000 nM, 200 nM, 40 nM and 8 nM,except for STIM003 which was used at 1000 nM, 200 nM, 40 nM, 8 nM and 2nM. Binding sensorgrams were double referenced using a buffer injection(i.e. 0 nM), and the data was fitted to the 1:1 model inherent to theProteOn XPR36 analysis software. The assay was run at 25° C. and usingHBS-EP as running buffer.

TABLE E7-1 Affinity and kinetic data for selected antibodies as measuredby SPR. Sample Ab Ligand ka kd KD (nM) STIM006 hICOS 6.67E+05 9.20E−0313.8 STIM003 hICOS 6.56E+05 8.62E−04 1.3 STIM001 hICOS 2.54E+04 1.12E−0344.0 STIM002 hICOS 3.20E+04 3.43E−02 1070.0 STIM006 mICOS 1.57E+035.00E−04 318.0 STIM003 mICOS 1.29E+06 5.03E−04 0.4 STIM001 mICOS5.66E+04 2.30E−02 407.0 STIM002 mICOS weak weak weak

In addition, a comparison was performed of antibody:antigen bindingaffinity at different pH values. As before, the dimeric human ICOSprotein, presented as the extracellular domain of ICOS fused to a humanFc region, was captured on the anti-human Fc capture surface createdusing the 3 antibody cocktail, immobilised on the GLC biosensor chip byprimary amine coupling. SPR analysis of recombinantly expressedanti-ICOS Fabs was carried out on the ProteOn XPR36 Array system(Biorad). The Fab fragments were used as analyte to generate bindingsensorgrams, which were double referenced with a buffer injection (i.e.,0 nM). The subsequent referenced sensorgrams were fitted to the 1:1model inherent to the ProteOn analysis software. Table E7-2 presentsaffinity and kinetic data for the antibodies, all run at 37° C. unlessstated, using either HBS-EP at pH 7.4/7.6 or pH 5.5 as indicated. Datawere fitted to the 1:1 model. Note that data for STIM002 fitted poorlyto the 1:1 model at both pH 7.4 and 5.5—the affinity for this antibodymay therefore be lower than indicated in the table.

TABLE E7-2 Relative affinity of STIM001, STIM002, STIM002-B and STIM003Fabs against recombinant human ICOS, at 37° C. except where stated.Antibody (Fab) pH ka kd KD (nM) STIM001 7.4 5.08E+04 3.23E−03 63.5 5.54.90E+04 3.58E−03 73.1 7.6 8.29E+04 3.54E−03 42.6 5.5 6.77E+04 5.41E−0380.3 7.6 (25° C.) 2.54E+04 1.12E−03 44 STIM002 7.4 3.72E+04 8.31E−03 2235.5 8.79E+04 3.67E−03 4.17 STIM002-B 7.4 8.28E+04 3.46E−03 41.8 5.58.64E+04 2.30E−03 26.6 STIM003 7.4 1.49E+06 2.54E−03 1.71 5.5 1.55E+061.58E−03 1.02 7.6 1.87E+06 3.70E−03 1.98 5.5 1.71E+06 1.94E−03 1.15 7.6(25° C.) 6.65E+06 0.862E−03  1.31

Comparison of the affinity data at different pH values indicated thatthe antibodies retain binding to their target across a physiological pHrange. The tumour microenvironment may be relatively acidic comparedwith blood, thus maintenance of affinity at low pH is a potentialadvantage in vivo to improve intra-tumoural T-reg depletion.

Example 8: Neutralisation of ICOS Ligand Binding to ICOS ReceptorAssayed by HTRF

Selected anti-ICOS antibodies were further assessed for their ability toneutralise ICOS ligand (B7-H2) binding to ICOS, using homogenous timeresolved fluorescence (HTRF). Human IgG1 and human IgG4.PE(null-effector) isotypes of the mAbs were assessed in:

-   -   HTRF assay for neutralisation of human B7-H2 binding to human        ICOS; and    -   HTRF assay for neutralisation of mouse B7-H2 binding to mouse        ICOS.        Anti-ICOS antibody C398.4A (hamster IgG in each case) was        included for comparison.

A number of antibodies were found to have high neutralising potency forhuman and/or mouse ICOS receptor-ligand binding, and the resultsindicated that some of these antibodies showed good cross-reactivity.The antibody isotype had no significant effect, differences in resultsbetween the IgG1 and IgG4.PE assays being within experimental error.

IgG1

In the human IgG1 assays, antibody C398.4A produced an IC50 of 1.2±0.30nM for the neutralisation of human ICOS ligand and an IC50 of 0.14±0.01nM for the neutralisation of mouse ICOS ligand.

IgG1 mAbs STIM001, STIM002, STIM003 and STIM005 produced similar IC50 toC398.4A using the human ICOS ligand neutralisation system and were alsocross-reactive, neutralising binding of mouse ICOS ligand to the mouseICOS receptor.

Two additional cross-reactive mAbs, STIM002-B and STIM004, showed weakerhuman and mouse ICOS ligand neutralisation.

STIM006, STIM007, STIM008 and STIM009 showed neutralisation of humanICOS ligand but did not demonstrate significant cross-reactivity in themouse ICOS ligand neutralisation system. Neutralising IC50 values formouse B7-H2 ligand could not be calculated for these antibodies.

TABLE E8-1 IC50 values for human IgG1 isotype mAb for neutralisation ofhuman ICOS Receptor binding to human B7-H2. See also FIG. 2. Mean IC50(nM) SD (nM) (n = 4) STIM001 2.2 1.3 STIM002 1.9 0.8 STIM002-B 3.6 3.5STIM003 1.3 0.5 STIM004 233 123 STIM005 2.5 0.8 STIM006 2.2 1.5 STIM0071.1 0.5 STIM008 1.6 1.4 STIM009 30.5 53 C398.4A 1.2 0.3

TABLE E8-2 IC50 values for human IgG1 isotype mAb for neutralisation ofmouse ICOS Receptor binding to mouse B7-H2. See also FIG. 3. Mean IC50(nM) SD (nM) (n = 3) STIM001 6.5 2.5 STIM002 6.9 2.1 STIM002-B 30 11.4STIM003 0.1 0 STIM004 22.1 15.4 STIM005 0.3 0.2 C398.4A 0.1 0IgG4.PE

As expected, IgG4.PE mAbs produced similar results to the IgG1 isotypes.

STIM001, STIM003 and STIM005 showed similar IC50 values to C398.4A usingthe human ICOS ligand neutralisation system. These mAbs were alsocross-reactive at neutralising mouse ICOS ligand. STIM002-B and STIM004produced weaker IC50 values for human ICOS B7-H2 neutralisation andmouse B7-H2 ligand. STIM007, STIM008 and STIM009 showed neutralisationof human ICOS ligand binding to human ICOS receptor but neutralisingIC50 values for mouse B7-H2 ligand could not be calculated in theseassays.

IgG4.PE isotypes of STIM006 and STIM002 were not assayed.

TABLE E8-3 IC50 values for human IgG4.PE isotype mAb for neutralisationof human ICOS Receptor binding to human B7-H2. See also FIG. 4. SD (nM)(n = 4 unless Mean IC50 (nM) otherwise stated) STIM001 1.3 0.2 STIM002-B3.4 1.8 STIM003 1.2 0.3 STIM004 161 152 (n = 3) STIM005 1.6 0.2 STIM0060.8 (n = 1) STIM007 0.8 0.1 STIM008 0.8 0.1 STIM009 4.6 2.2 C398.4A 2.83.8

TABLE E8-4 IC50 values for human IgG4.PE isotype mAb for neutralisationof mouse ICOS Receptor binding to mouse B7-H2. See also FIG. 5. MeanIC50 (nM) SD (nM) (n = 3) STIM001 4.7 2.1 STIM002-B 43.9 25.7 STIM0030.2 0.1 STIM004 30 14 STIM005 0.3 0.1 C398.4A 0.2 0.1Materials and Methods

Test antibodies and isotype controls were diluted in assay buffer (0.53M Potassium Fluoride (KF), 0.1% Bovine Serum Albumin (BSA) in 1×PBS)from a starting working concentration of up to 4 μM, 1 μM final to 0.002nM, 5.64e−4 nM final over 11 point titration, 1 in 3 dilutions.Titrations of 5 μl of antibody were added to 384w white walled assayplate (Greiner Bio-One). Positive control wells received 5 μl of assaybuffer only.

5 μl of ICOS receptor (human ICOS-mFc, 20 nM, 5 nM final or mouseICOS-mFc 4 nM, 1 nM final (Chimerigen)) was added to required wells.Plate was incubated for 1 hr at room temperature (RT). After incubation,5 μl of either mouse or human ICOS ligand, (B7-H2, R&D Systems)conjugated to Alexa 647 (Innova Bioscience) was diluted to either 32 nM(8 nM final) for human B7-H2 or 30 nM, 7.5 nM final for mouse B7-H2 andadded to all wells of assay plate except negative control wells whichinstead received 5 μl of assay buffer.

Finally, 5 μl of anti-mouse IgG donor mAb (Southern Biotech) labelledwith europium cryptate (Cis Bio), 40 nM, 10 nM final was added to eachwell and the assay was left in the dark at RT to incubate for a further2 hours. After incubation, assay was read on Envision plate reader(Perkin Elmer) using a standard HTRF protocol. 620 nm and 665 nm channelvalues were exported to Microsoft Excel (Microsoft) and % Delta-F and %Neutralisation calculations performed. Titration curves and IC50 values[M] were plotted using Graphpad (Prism). IC50 values were calculated byfirst transforming the data using equation X=Log(X). The transformeddata was then fitted using nonlinear regression, using fittingalgorithm, log (inhibitor) vs. response—variable slope (fourparameters).

% Delta-F Calculation:

665/620 nm ratio for ratio metric data reduction.

${\%\mspace{14mu}{Delta}\mspace{14mu} F} = {\frac{\left( {{{665/620}\mspace{14mu}{nm}\mspace{14mu}{Well}\mspace{14mu}{Signal}\mspace{14mu}{Ratio}} - {{Signal}\mspace{14mu}{Negative}\mspace{14mu}{Control}}} \right)}{\left( {{Signal}\mspace{14mu}{Negative}\mspace{14mu}{Control}} \right)}*100}$  Signal  Negative  control = average  of  minimum  signal  ratio.% Neutralisation:

${\%\mspace{14mu}{Max}\mspace{14mu}({neutralisation})} = {\frac{\left( {{\%\mspace{14mu}{Delta}\text{-}F\mspace{14mu}{of}\mspace{14mu}{sample}\mspace{14mu}{well}} - {{Negative}\mspace{14mu}{Control}}} \right)}{\left( {{{Positive}\mspace{14mu}{Control}} - {{Negative}\mspace{14mu}{Control}}} \right)}*100}$

Example 9a: T-Cell Activation

STIM001 and STIM003 agonistic potential on cytokine production wastested in plate-bound and soluble format in a human primary T-cellactivation assay where anti-CD3 and anti-CD28 Abs were addedconcurrently to the anti-ICOS Ab to induce ICOS expression on effectorT-cells. Effect of the ICOS co-stimulation on the level of IFNγ producedby these activated T-cells were assessed using ELISA at 72 hrspost-activation.

Materials and Methods

T-Cell Activation Assay 1:

Isolation of Mononuclear Cells from Human Peripheral Blood (PBMC):

Leukocyte cones were collected from healthy donors and their content wasdiluted up to 50 ml with phosphate buffered saline (PBS, from Gibco) andlayered into 2 centrifuge tubes on top of 15 mL Ficoll-Paque (from GEHealthcare). PBMC were separated by density gradient centrifugation (400g for 40 min without brake), transfered to a clean centrifuge tube andthen washed with 50 mL PBS, twice by centrifuging at 300 g for 5 min andtwice by centrifuging at 200 g for 5 min. PBMC were then resuspended inR10 media (RPMI+10% heat-inactivated Fetal Bovine Serum, both fromGibco) and their cell count and viability assess with EVE™ AutomatedCell Counter (from NanoEnTek).

ICOS Antibodies (Abs) Preparation and Dilutions:

STIM001 and STIM003 were tested in 3 formats: plate-bound, soluble orsoluble plus F(ab′)₂ Fragments (109-006-170 from Jackson ImmunoResearch) which crosslink the anti-ICOS Abs.

For plate-bound format: the anti-ICOS Abs and their isotype control wereserially diluted 1:3 in PBS to give final antibody concentrationsranging from 45 to 0.19 μg/mL. 100 μL of diluted antibodies were coatedin duplicate into a 96-well, high-binding, flat-bottom plate (CorningEIA/RIA plate) overnight at 4° C. Plate was then washed with PBS and 125μl of R10 were added to each well.

For soluble format: The anti-ICOS Abs and their isotype control wereserially diluted 1:3 in R10 media to give an 2× Ab stock concentrationsranging from 90 to 0.38 μg/mL. 125 μl of diluted Abs were pipetted induplicate into a 96-well, flat-bottom plate.

For crosslinked soluble format: The anti-ICOS Abs and their isotypecontrol were mixed with F(ab′)₂ Fragments at 1M to 1M ratio. Abs/F(ab′)₂Fragments mixes were then 1:3 serially diluted in R10 media to give an2× Ab concentrations ranging from 90 to 0.38 μg/mL for ICOS and from 60to 0.24 μg/ml for F(ab′)₂ Fragments. 125 μl of diluted Abs were pipettedin duplicate into a 96-well, flat-bottom plate.

T-Cell Isolation, Cultures and IFN-γ Quantification:

T-cell were negatively isolated from PBMC using the EasySep Human T CellIsolation Kit (from Stemcell Technologies) and resuspended at 2×10⁶/mLin R10 media supplemented with 40 μl/ml of Dynabeads Human T-ActivatorCD3/CD28 (from Life Technologies).

125 μl of T-cell suspension were added to Ab-containing plates to give afinal cell concentration of 1×10⁶ cells/ml and cultured for 72 hrs at37° C. and 5% CO₂. Cell free supernatants were then collected and keptat −20° C. until analysis of secreted IFNγ by ELISA (duoset kit fromR&D).

This experiment was repeated on T-cells isolated from 6 independentdonors and 2 technical replicates were included for each assaycondition.

T-Cell Activation Assay 2 (STIM-REST-STIM Assay):

STIM001 and STIM003 agonist potential on cytokine release were alsotested plate-bound in a human T-cell assay where T-cells wereprestimulated by anti-CD3 and anti-CD28 Abs for 3-days to induce ICOSexpression before being rested for 3-days to reduce their activationlevels. ICOS expression was confirmed by FACS staining after stimulation(Day 3) and resting (Day 6). These stimulated rested T-cells were thencultured with STIM001 or STIM003 in presence or absence of CD3 Ab toassess the requirement of TCR engagement. Effects of the ICOSco-stimulation were assessed after 72 hrs on the levels of IFNγ, TNFαand IL-2 present in the culture.

ICOS Abs Dilutions and Coating:

Anti-human CD3 (clone UCHT1 from eBioscience) was diluted in PBS to a 2×Ab concentration of 10 μg/mL. 50 μl of PBS or 50 μl of diluted CD3 Abwere pipetted into a 96-well, high-binding, flat-bottom plate. STIM001,STIM003 and their isotype control were 1:2 serially diluted in PBS togive final 2× antibody concentrations ranging from 20 to 0.62 μg/mL. 50μL of diluted anti-ICOS Ab were added to wells containing either PBS (noTCR engagement) or diluted CD3 Ab (TCR engagement). Plates were coatedovernight at 4° C.

T-Cell Isolation, Cultures and IFN-γ Quantification:

PBMC from leukocyte cones were obtained as described in T-cellactivation assay 1. T-cell were negatively isolated from this PBMC usingthe EasySep Human T Cell Isolation Kit (from Stemcell Technologies).T-cells were resuspended at 1×10⁶/ml in R10 media supplemented with 20μl/mL of Dynabeads Human T-Activator CD3/CD28 (from Life Technologies)and cultured for 3-days at 37° C. and 5% CO₂ (Stimulation). At day 3dynabeads were removed from the culture. T-cells were then washed (300 gfor 5 min), counted and resuspended at 1.5×10⁶/ml in R10 media andculture at 37° C. and 5% CO₂ for 3-more days (Resting phase).

At day 6 stimulated rested T-cells were then washed (300 g for 5 min),counted and resuspended at 1×10⁶/mL in R10 media and 250 μl of T-cellsuspension were added to ICOS Ab-coated plates and cultured for 72 hrsat 37° C. and 5% CO₂. Cell free supernatants were then collected andkept at −20° C. until analysis of secreted cytokines on the MSDplatform. This experiment was repeated with T-cells isolated from 5independent donors and 3 technical replicates were included for eachassay condition.

Results

Both STIM001 and STIM003 tested positive for inducing IFNγ expressiontherefore demonstrating agonism in both assays.

Example 9b: T Cell Activation Assay 1 Data

T cell activation assay 1 was performed as described in Example 9a,using T cells isolated from 8 independent donors, testing each ofSTIM001 and STIM003 in human IgG1 format. Hamster anti-ICOS antibodyC398.4A and a hamster antibody isotype control were included forcomparison. 2 technical replicates were included for each assaycondition.

Results are shown in FIG. 16, FIG. 17 and FIG. 18. As noted before, bothSTIM001 and STIM003 tested positive for inducing IFNγ expressiontherefore demonstrating an agonistic effect on human primary T cells.

Cross-linked antibodies acted as agonists of T cell activation, asindicated by the strong enhancement of IFNγ induction in the presence ofthe Fc-linking F(ab′)₂ fragments, compared with either soluble antibodyor with control. IFNγ expression in the T cells increased withincreasing concentration of cross-linked STIM001 or STIM003 (FIG. 16,lower panels). Agonism was also observed for both STIM001 and STIM003 inplate-bound form and, more weakly, for the hamster antibody C398.4A, asindicated by the increase in IFNγ expression observed in the T cellswith increasing concentration of antibody (FIG. 16, top panels).

Magnitude of the IFNγ response varied between T cells obtained fromdifferent donors, but STIM001 consistently produced an increase in IFNγexpression in T cells compared with IFNγ expression observed withcontrol antibody (HC IgG1). When considering data from assays with Tcells from all 8 donors, it is seen that treatment of T cells withSTIM001 significantly increased IFNγ expression compared with treatmentwith isotype control antibody, in plate-bound form, soluble form andcross-linked form (FIG. 17). STIM001 thus behaved as an agonist of Tcell activation in all three formats.

Similar effects were observed with STIM003 (FIG. 18). Levels of IFNγinduced by STIM003 hIgG1 were compared with levels of IFNγ induced byits isotype control (HC IgG1) at a given dose of antibody in the assay,for 8 independent donors. Despite the variability between donors, themean increase in IFNγ level induced by STIM003 was significant whencompared against HC IgG1. It is proposed that STIM001, and the otherSTIM antibodies described here, have the potential to similarly promoteT cell activation in vivo. As discussed previously, agonism of activatedICOS-expressing T cells may be mediated by the anti-ICOS antibodybinding to and inducing multimerisation of the ICOS receptor on the Tcell surface. Example 9c: T cell activation assay 2 data

T cell activation assay 2 was performed as described in Example 9a.

In the absence of TCR engagement (no anti-CD3 antibody), levels ofcytokines produced from the primary T cells were low and no increase wasinduced by STIM001 (hIgG1), STIM003 (hIgG1) or antibody C398.4A even atthe highest concentration of 10 μg/ml. In contrast, when the anti-ICOSantibodies were added to T cells in combination with the anti-CD3antibody, each of STIM001 (hIgG1), STIM003 (hIgG1) and C398.4A showed adose-dependent trend to increase expression of IFNγ, TNFα and, to alesser degree, IL-2.

Data from primary T cells treated with anti-ICOS antibodies underconditions of TCR engagement are shown in FIG. 19. Although markedincreases in cytokine expression were observed for each of STIM001,STIM003 and C389.4A relative to their respective isotype controls, thedifference did not reach statistical significance in this assay. Furtherreplicates of the assay with responsive primary T cells from more donorswould be expected to generate statistically significant results.

Example 10a: ADCC Assay

STIM001 and STIM003 potential to kill via ADCC was tested in the DelfiaBATDA cytotoxicity assay (Perkin Elmer) using human primary NK cells aseffector and ICOS high MJ cell line (ATCC, CRL-8294) as target cells. MJcells are human CD4 T-lymphocyte cells that express high levels of ICOSprotein.

This method is based on loading target cells with an acetoxymethyl esterof fluorescence enhancing ligand (BATDA) which quickly penetrates thecell membrane. Within the cell the ester bonds are hydrolysed to form ahydrophilic ligand (TDA) which no longer passes the membrane. Aftercytolysis the ligand is released and can be detected by addition ofEuropium which forms with the BATDA a highly fluorescent and stablechelate (EuTDA). The measured signal correlates directly with the amountof lysed cells.

Materials and Methods

Target Cell Labelling:

According to the manufacturer's instructions, MJ cells were resuspendedat 1×10⁶/mL in assay media (RPMI+10% ultra-low IgG FBS, from Gibco) andloaded with 5 μl/mL of Delfia BATDA reagent (Perkin Elmer) for 30 min at37° C. MJ cells were then washed 3 times with 50 mL PBS (300 g for 5min) and resuspended at 8×10⁵/ml in assay media supplemented with 2 mMProbenecid (from Life technologies) to reduce BATDA spontaneous releasefrom the cells.

ICOS Ab Dilution:

STIM001, STIM003 and their isotype control were 1:4 serially diluted inassay media+2 mM Probenecid to give final 4× antibody concentrationsacross a range down to 80 pg/mL.

NK-Cell Isolation and Culture:

PBMC from leukocyte cones were obtained as described in T-cellactivation assay 1. NK-cell were negatively isolated from this PBMCusing the EasySep Human NK Cell Isolation Kit (from StemcellTechnologies) and resuspended at 4×10⁶/ml in R10 media+2 mM Probenecid.NK cell purity was checked to be above 90% by staining for CD3−/CD56+.

50 μl of diluted Ab, 50 μl of BATDA loaded MJ cells, 50 μl of NK cellsand 50 μl of assay media+2 mM Probenecid (final volume of 200 μl/well)were added in each well to give a final Ab concentration across a rangedownto 20 pg/mL and an effector:target ratio of 5:1. Wells containing MJcells only or MJ cells+delfia lysis buffer (Perkin Elmer) are used todetermine spontaneous and 100% BATDA release.

The assay was run at 37° C., 5% CO₂ for 2 hrs before transferring 50 μlof cell-free supernatant into a DELFIA Microtitration Plates (PerkinElmer). 200 μl of Delfia Europium solution (Perkin Elmer) was added tothe supernatants and incubated for 15 min at Room Temperature.Fluorescent signal was then quantified with EnVision Multilabel Reader(PerkinElmer).

Specific release induced by STIM001 and STIM003 was calculated accordingto the kit instructions. This experiment was repeated with NK-cells fromindependent donors and 3 technical replicates were included for eachassay condition.

Results

Anti-ICOS antibodies STIM001 (hIgG1) and STIM003 (hIgG1) kill ICOSpositive human MJ cells in a primary NK dependent ADCC assay (2 hourtime point). See also FIG. 6a . Sub-Nanomolar EC50 were achieved in thisassay for both molecules tested.

TABLE E10-1 EC50 (Molar unit) of STIM001 in the NK primary cells ADCCassay from 2 donors (2 hour time point). EC50 Donor 1 Donor 2 STIM0011.21e−10 5.29e−10

Example 10b: ADCC Assay with MJ Target Cells

The experiment was performed according to the Materials and Methods setout in Example 10a. STIM001, STIM003 and isotype control were 1:4serially diluted in assay media+2 mM Probenecid to give final 4×antibody concentrations ranging from 40 μg/mL to 80 pg/mL. 50 μl ofdiluted Ab, 50 μl of BATDA loaded MJ cells, 50 μl of NK cells and 50 μlof assay media+2 mM Probenecid (final volume of 200 μl/well) were addedin each well to give a final Ab concentration ranging from 10 μg/mL to20 pg/mL and an effector:target ratio of 5:1.

Results are shown in FIG. 6 (b-d) and in the table below. STIM001(hIgG1) and STIM003 (hIgG1) killed ICOS positive human MJ cells in theprimary NK dependent ADCC assay, measured at the two hour time point.

TABLE E10-2 EC50 (Molar unit) of STIM001 and STIM003 in the NK primarycell ADCC assay from 3 donors (2 hour time point). EC50 Donor 1 Donor 2Donor 3 STIM001 1.21e−10  5.29e−10 (0.529 nM) 2.92e−09 (2.92 nM) (0.121nM) STIM003 2.33e−12 3.58-e−11 (35.8 pM) 1.01e−10 (0.101 nM) (2.33 pM)

Example 10c: ADCC Assay with ICOS-Transfected CCRF-CEM Target Cells

STIM001 and STIM003 hIgG1 potential to kill via ADCC was further testedin the Delfia BATDA cytotoxicity assay (Perkin Elmer) using humanprimary NK cells as effector and ICOS-transfected CCRF-CEM cells (ATCC,CRL-119) as target cells. CCRF-CEM is a human T lymphoblast line,originating from peripheral blood from a patient with acutelymphoblastic leukaemia. Antibody-mediated killing of CCRF-CEM cells wasconfirmed for both STIM001 and STIM003 in this assay.

Materials and Methods

Materials and Methods were as set out in Example 10a, but using CCRF-CEMcells obtained from ATCC (ATCC CCL-119) rather than MJ cells as thetarget cells, and using an incubation time of 4 hours.

CCRF-CEM cells were transfected with ICOS. A synthetic string DNAencoding full length human ICOS (with signal peptide, as shown in theappended sequence listing), codon-optimised for mammalian expression,was cloned into an expression vector under control of the CMV promoterand flanked by 3′ and 5′ piggyBac specific terminal repeat sequencesfacilitating stable integration into the cell genome (see [40]). Theexpression vector contained a puromycin selection cassette to facilitatestable cell line generation. The human ICOS expression plasmid wasco-transfected with a plasmid encoding piggyBac transposase into CEMCCRF cells by electroporation. 24 hours after transfection, the mediawas supplemented with puromycin and grown for at least two weeks toselect stable cell lines, with media being exchanged every 3-4 days. Theexpression of human ICOS was assessed by flow cytometry using ananti-human ICOS-PE conjugated antibody (eBioscience). Complete CEM mediawas made up of Advanced RPMI Medium containing 10% (v/v) FBS and 2 mMGlutamax.

STIM001 (hIgG1), STIM003 (hIgG1) and an isotype control antibody (HCIgG1) were serially diluted in assay media to give final 4× antibodyconcentrations ranging from 20 μg/mL to 80 pg/mL.

50 μl of diluted Ab, 50 μl of BATDA loaded ICOS-transfected CEM cells,50 μl of NK cells and 50 μl of assay media (final volume of 200 μl/well)were added in each well to give a final Ab concentration ranging from 5μg/mL to 20 pg/mL and an effector:target ratio of 5:1.

Results

STIM001 (hIgG1) and STIM003 (hIgG1) killed ICOS-transfected CCRF-CEMcells in the primary NK dependent ADCC assay, measured at the four hourtime point. Results are shown in FIG. 6 (e-g) and in the table below.

TABLE E10-3 EC50 (Molar unit) of STIM001 and STIM003 in the NK primarycell ADCC assay from 3 donors (4 hour time point). EC50 Donor 4 Donor 5Donor 6 STIM001 3.92e−12 3.95e−12 (3.95 pM) 3.75e−12 (3.75 pM) (3.92 pM)STIM003 Approx 3 pM* 8.95e−13 (0.895 pM) 1.03e−12 (1.03 pM) *Valueestimated from incomplete curve.

Example 11a: CT26 Syngeneic Model

Improved anti-tumour in vivo efficacy was shown in a CT26 syngeneicmodel by combining anti-ICOS (STIM001 mIgG2a, effector enable) withanti-PDL1 (10F9G2).

Materials and Methods

Efficacy studies were performed in Balb/c mice using the sub-cutaneousCT26 colon carcinoma model (ATCC, CRL-2638). This model is poorlysensitive to PD1/PDL1 blockade and only tumour growth delay (no stabledisease or cure) is usually observed in response to 10F9.G2 (anti-PDL1)and RMT1-14 (anti-PD1) monotherapies. Therefore this model constitutes arelevant model for looking at anti-PD1, anti-PDL1 intrinsic resistancefor combination studies. All in vivo experiments were performed inaccordance with the UK Animal (Scientific Procedures) Act 1986 and theEU Directive 86/609, under a UK Home Office Project Licence and approvedby the Babraham Institute Animal Welfare and Ethical Review Body.

Balb/c mice were supplied by Charles River UK at 6-8 weeks of ageand >18 g and housed under specific pathogen-free conditions. A total of1×10⁵ CT26 cells (passage number below P20) were subcutaneously injectedinto the left flanks of mice. Unless stated otherwise, treatment wereinitiated at day 6 post tumour cells injection. The CT26 cells werepassaged in vitro by using Accutase (Sigma), washed twice in PBS andresuspended in RPMI supplemented with 10% fetal calf serum. Cellviability was confirmed to be above 90% at the time of tumour cellinjection.

For in vivo studies STIM001 anti-ICOS agonist (cross reactive to mouseICOS protein) was reformatted as mouse IgG1 and mouse IgG2a to test theas effector function null and as effector function enable, respectively.The Anti-PDL1 was sourced from Biolegend (Cat.no:124325). The hybridcontrols were generated in Kymab (mIgG2a isotype) or from commercialsource (hamster isotype HTK888, Biolegend (Part No92257, Lot B215504)).All antibodies were dosed intraperitoneal (IP) at 10 mg/kg (1 mg/ml in0.9% saline) three times a week from day 6 (dosing for 2 weeks day 6-18)as monotherapies or by combining anti-PDL1 with anti-ICOS antibodies.Animal weight and tumour volume were measured 3 times per week from theday of tumour cell injection. Tumour volume was calculated by use of themodified ellipsoid formula 1/2(Length×Width2). Mice were kept on studiesuntil their tumour reached an average diameter of 12 mm³ or, in rarecase, when incidence of tumour ulceration was observed (welfare). Theexperiment was stopped at day 50. The human endpoint survival statisticswere calculated using the Kaplan-Meier method with Prism. This approachwas used to determine if specific treatments were associated withimproved survival.

TABLE E11-1 Treatment groups Number of Groups animals Treatments (T.I.W,IP from day 6) 1 10 10 mg/kg mIgG2a and 10 mg/kg IgG isotype Control(HTK888) 2 10 10 mg/kg Anti-ICOS STIM001 mIgG1 3 10 10 mg/kg Anti-ICOSSTIM001 mIgG2a 4 10 10 mg/kg Anti-PD-L1 (10F9.G2) 5 10 10 mg/kganti-PD-L1 plus 10 mg/kg Anti-ICOS STIM001 mIgG1 6 10 10 mg/kganti-PD-L1 plus 10 mg/kg Anti-ICOS STIM001 mIgG2aResults

As shown in FIG. 7, FIG. 8 and FIG. 9, ICOS agonists can delay diseaseprogression and cure a proportion of animals from the CT-26 subcutaneoustumours either as a monotherapy or in combination with anti-PDL1.Anti-PDL1 monotherapy induced tumour growth delay but no stable diseaseor curative potential was observed. The combination was more effectiveat treating the tumours than the anti-ICOS monotherapies. This studyalso highlighted that STIM001 in the mouse IgG2a format (effectorfunction enable) was more potent than the mouse IgG1 (effector null)format at triggering an anti-tumour response in this model.

Example 11b: Strong Anti-Tumour Efficacy In Vivo in CT26 Syngeneic Modelfor Combination of Anti-ICOS mIgG2a with Anti-PDL1 mIgG2a

An in vivo combination study was performed with STIM001 with a mousecross reactive anti-human PDL1 antibody designated AbW. For this in vivowork, STIM001 was reformatted as mouse IgG1 and mouse IgG2a to compareits efficacy with low effector function or as effector function enabledmolecule, respectively. The anti-PDL1 AbW was generated in the sameformats (mouse IgG1 and mouse IgG2A).

The efficacy studies were performed in Balb/c mice using thesub-cutaneous CT26 colon carcinoma model (ATCC, CRL-2638). Balb/c micewere supplied by Charles River UK at 6-8 weeks of age and >18 g andhoused under specific pathogen-free conditions. A total of 1×10E5 CT26cells (passage number below P20) were subcutaneously injected into theright flanks of mice. Unless stated otherwise, treatment were initiatedat day 6 post tumour cells injection. The CT26 cells were passaged invitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice inPBS and resuspended in RPMI supplemented with 10% foetal calf serum.Cell viability was confirmed to be above 90% at the time of tumour cellinjection.

STIM001 and anti-PDL1 antibodies were dosed concurrently in combinationsintraperitoneal (IP) at 200 μg each (1 mg/ml in 0.9% saline) three timesa week from day 6 (dosing for 2 weeks between day 6-17) post tumour cellimplantation. Tumour growth was monitored and compared to tumours ofanimals treated with a mixture of isotype control antibodies (mIgG1 andmIgG2A). Animal weight and tumour volume were measured 3 times a weekfrom the day of tumour cell injection. Tumour volume was calculated byuse of the modified ellipsoid formula 1/2(Length×Width2). Mice were kepton studies until their tumour reached an average diameter of 12 mm3 or,in rare case, when incidence of tumour ulceration was observed(welfare). The experiment was stopped at day 60. The human endpointsurvival statistics were calculated using the Kaplan-Meier method withPrism. This approach was used to determine if specific treatments wereassociated with improved survival.

TABLE E11-2 Treatment groups for STIM001 2 × 2 combinations Number ofGroup animals Treatment regimen (3 time a week for 2 weeks) 1 10mIgG2a + mIgG1 isotypes 200 μg each 2 10 Anti-ICOS mIgG1 STIM001 +Anti-PD-L1 mIGg1 (AbW) 200 μg each 3 10 Anti-ICOS mIgG2a STIM001 +Anti-PD-L1 mIGg2a (AbW) 200 μg each 4 10 Anti-ICOS mIgG2a STIM001 +Anti-PD-L1 mIGg1 (AbW) 200 μg each 5 10 Anti-ICOS mIgG1 STIM001 +Anti-PD-L1 mIGg2a (AbW) 200 μg each

Results are shown in FIG. 10. All antibody combinations delayed tumourgrowth and extended the survival (time to reach human endpoint) oftreated animals when compared to isotype control-treated animals.Interestingly, when combined with anti-PDL1 (independently of itsformat, mIgG1 or mIgG2a), STIM001 mIgG2a antibody was more effective atinhibiting tumour growth than STIM001 in the mIgG1 format. These datasuggest the advantage of an anti-ICOS antibody having effector functionto maximize anti-tumour efficacy. Notably, STIM001 mIgG2a in combinationwith aPD-L1 mIgG2a demonstrated the strongest anti-tumour efficacy andimproved survival (90% of animals to show response and 60% were curedfrom the disease at day 60).

Similarly, STIM003 mIgG1 and mIgG2a were tested as monotherapy or incombination with anti-PDL1 (AbW) mIgG2a in the same CT26 tumour models.STIM003 and anti-PDL1 antibodies were dosed in animals as monotherapy orin combination by intraperitoneal injection (IP) of 200 μg of antibodieseach (1 mg/ml in 0.9% saline) three times a week from day 6 (dosing for2 weeks between day 6-17) post tumour cell implantation. In thisexperiment tumour sizes were monitored for 41 days. The human endpointsurvival statistics were calculated using the Kaplan-Meier method withPrism. This approach was used to determine if specific treatments wereassociated with improved survival.

TABLE E11-3 Treatment groups for STIM003 combination with anti-PDL1 AbWIgG2a Number of Treatment regimen Group animals (3 times a week for 2weeks from day 6) 1 10 mIgG2a + mIgG1 isotypes control 200 μg each 2 10Anti-PD-L1 mIgG2a (AbW) 200 μg 3 10 STIM003 mIgG1 200 μg 4 10 STIM003mIgG2a 200 μg 5 10 STIM003 mIgG1 + Anti-PD-L1 mIGg2a (AbW) 200 μg each 610 STIM003 mIgG2a + Anti-PD-L1 mIGg2a (AbW) 200 μg each

Results are shown in FIG. 11. Monotherapies using aPDL1 (AbW) andSTIM003 mIgG2a demonstrated mild anti-tumour activity (one animal wascured of the disease in each group). Combinations of STIM003 mIgG1 ormIgG2a with aPDL1 (AbW) mIgG2a showed strong anti-tumour efficacy.Interestingly, by day 41, when combined with aPDL1 mIgG2a, STIM003mIgG2a was more potent at inhibiting tumour growth than STIM003 mIgG1(60% vs 30% of animals cured of the disease, respectively). The datafurther highlighted the advantage of an effector format for anti-ICOSantibodies to maximize anti-tumour efficacy.

Altogether, these data demonstrate that combination of an anti-ICOSantibody STIM001 or STIM003 with anti-PDL1 results in the strongestanti-tumour response when both antibodies have an effector enabledfunction. Suitable corresponding human antibody isotypes would includehuman IgG1, optionally with further enhanced effector function e.g.,afucosylated IgG1.

Kaplan Meier curves for mice treated with the combination of anti-PDL1mIgG2a and STIM003 mIgG2a and for each agent individually are shown inFIG. 29.

Example 11c: Single Dose of STIM003 Antibody Resets the TumourMicroenvironment (TME) and Results in Strong Anti-Tumour Efficacy inCombination with Continuous Anti-PD-L1 Dosing

This study compared single vs multiple dosing of STIM003 mIgG2A togetherwith multiple dosing of anti-PDL1 antibody (AbW). The data indicate thata single dose of anti-ICOS antibody could alter the tumourmicroenvironment so as to allow an anti-PD-L1 antibody to exert agreater effect. This can be envisaged as a “resetting” of the TME by theanti-ICOS antibody.

As before, these efficacy studies were performed in Balb/c mice usingthe sub-cutaneous CT26 colon carcinoma model (ATCC, CRL-2638). Balb/cmice were supplied by Charles River UK at 6-8 weeks of age and >18 g andhoused under specific pathogen-free conditions. A total of 1×10E5 CT26cells (passage number below P20) were subcutaneously injected into theright flanks of mice. Unless stated otherwise, treatments were initiatedat day 6 post tumour cells injection. The CT26 cells were passaged invitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice inPBS and resuspended in RPMI supplemented with 10% foetal calf serum.Cell viability was confirmed to be above 90% at the time of tumour cellinjection.

Treatment groups are shown in Table E11-4. STIM003 and anti-PDL1antibodies were dosed intraperitoneal (IP) at 10 mg/kg (1 mg/ml in 0.9%saline). Treatments were initiated from day 6 post tumour cellimplantation. Tumour growth was monitored and compared with tumours ofanimals treated with saline. Animal weight and tumour volume weremeasured 3 times a week from the day of tumour cell injection. Tumourvolume was calculated by use of the modified ellipsoid formula1/2(Length×Width2). Mice were kept on studies until their tumour reachedan average diameter of 12 mm³ or, in rare case, when incidence of tumourulceration was observed (welfare). The experiment was stopped at day 55.

Data are shown in FIG. 34. Concurrent dosing of STIM003 and anti-PDL1for 6 doses from day 6 resulted in a strong anti-tumour efficacy in theCT26 model with 5/8 animals being tumour free at the end of the study(day 55). Interestingly, similar anti-tumour efficacy was achieved witha single dose of STIM003 followed by multiple dose of anti-PDL1 asmonotherapy. When combined with anti-PD-L1 mIgG2a, similar overallefficacy was observed between dosing STIM003 once (C) vs dosing 6 times(B). When compared with saline treated group (A) where only one animalhad a spontaneous tumour rejection (rare in this model), the groupstreated with combined drugs had full tumour rejection in 62.5% of theanimals by the end of the experiment (day 55). The data suggest that theSTIM003 antibody could be used to reset the tumour microenvironment andthat the antibody allows immune-checkpoint resistant tumours to becomesensitive to anti-PDL1. As previously shown (Example 11b), the CT26tumour cell line is not strongly responsive to anti-PDL1 monotherapy. Itappears that STIM003 causes changes that facilitate anti-tumour activityof the anti-PDL1 therapy.

Example 12: Antibody Sequence Analysis

Framework regions of antibodies STIM001, STIM002, STIM002-B, STIM003,STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 were comparedwith human germline gene segments to identify the closest match. SeeTable E12-1 and Table E12-2.

TABLE E12-1 Heavy chain germline gene segments of anti-ICOS Abs Heavychain V D J STIM001 IGHV1-18*01 IGHD6-19*01 IGHJ6*02 STIM002 IGHV1-18*01IGHD3-10*01 IGHJ6*02 STIM002-B IGHV1-18*01 IGHD3-10*01 IGHJ6*02 STIM003IGHV3-20*d01 IGHD3-10*01 IGHJ4*02 STIM004 IGHV3-20*d01 IGHD3-10*01IGHJ4*02 STIM005 IGHV1-18*01 IGHD3-9*01 IGHJ3*02 STIM006 IGHV3-11*01IGHD3-10*01 IGHJ6*02 STIM007 IGHV2-5*10 IGHD3-10*01 IGHJ6*02 STIM008IGHV2-5*10 IGHD3-10*01 IGHJ6*02 STIM009 IGHV3-11*01 IGHD3-9*01 IGHJ6*02

TABLE E12-2 Kappa light chain germline gene segments of anti-ICOS AbsLight chain V J STIM001 IGKV2-28*01 IGKJ4*01 STIM002 IGKV2-28*01IGKJ2*04 STIM002-B IGKV2-28*01 IGKJ2*04 STIM003 IGKV3-20*01 IGKJ3*01STIM004 IGKV3-20*01 IGKJ3*01 STIM005 IGKV1D-39*01 IGKJ1*01 STIM006IGKV2-28*01 IGKJ2*04 STIM007 IGKV3-11*01 IGKJ4*01 STIM008 IGKV3-11*01IGKJ4*01 STIM009 IGKV2-28*01 IGKJ1*01

Additional antibody sequences were obtained by next generationsequencing of PCR-amplified antibody DNA from further ICOS-specificcells that were sorted from the immunised mice as described in Example3. This identified a number of antibodies that could be grouped intoclusters with STIM001, STIM002 or STIM003 based their heavy and lightchain v and j gene segments and CDR3 length. CL-61091 clustered withSTIM001; CL-64536, CL-64837, CL-64841 and CL-64912 clustered withSTIM002; and CL-71642 and CL-74570 clustered with STIM003. Sequencealignments of the antibody VH and VL domains are shown in FIGS. 35 to37.

TABLE E12-3 Antibodies clustered by sequence. ANTIBODIES VH_V_GENEVH_J_GENE VH_CDR3_NT_LENGTH VL_V_GENE VL_J_GENE VL_CDR3_NT_LENGTHSTIM001, 1-18 6 42 2-28 4 27 CL-61091 STIM002, CL- 1-18 6 51 2-28 2 2764536, CL-64837, CL-64841, CL-64912 STIM003, CL- 3-20 4 51 3-20 3 2771642, CL-74570 STIM004 3-20 4 51 3-20 3 24 STIM005 1-18 3 51 1D-39 1 24STIM006 3-11 6 63 2-28 2 30 STIM007, STIM008 2-5  6 48 3-11 4 27 STIM0093-11 6 60 2-28 1 27

Example 13: Monism of ICOS-Expressing MJ Cells by Bead-Bound Antibody

Antibodies STIM001, STIM002 and STIM003, the anti-ICOS antibody C398.4A,and ICOS ligand (ICOSL-Fc), were each covalently coupled to beads andassessed for their ability to induce expression of the cytokine IFN-γfrom MJ cells grown in culture. Human IgG1 and Clone C398.4A isotypecontrols coupled to beads were assessed in parallel.

Data are shown in FIG. 12 and Table E13 below.

Each of the anti-ICOS antibodies demonstrated agonism in this assay,stimulating MJ cells as determined by IFN-γ quantification significantlyabove that observed by their cognate isotype controls within the dynamicrange of the assay.

STIM003 and Clone C398.4A produced lower top asymptote values (95% CI:3.79 to 5.13 and 3.07 to 4.22, respectively) but more potent Log EC50values (95% CI: −9.40 to −9.11 and −9.56 to −9.23, respectively)compared with STIM001 (Top 95% CI: 7.21 to 8.88 and Log EC50 95% CI:−8.82 to −8.63) and STIM002 (Top 95% CI: 5.38 to 6.95 and Log EC50 95%CI: −9.00 to −8.74). Because incomplete curves (Top out of dynamic rangeof assay) were produced for ICOSL-Fc and Clone C398.4A isotype control,the fitted Top and Log EC50 values were not treated as reliable. HumanIgG1 Hybrid Control produced a complete curve, however the area underthe curve was not significantly different from 0 and it was thereforenot deemed to be an agonist.

TABLE E13 Summary table of bead bound MJ cell in vitro activation assay.Best-fit Human IgG1 Clone values STIM001- STIM002- STIM003- HybridControl- C398.4A- Clone C398.4AIC- 95% CI beads beads beads beads beadsbeads ICOSL-Fc-beads Bottom −0.24 to 0.36   −0.38 to 0.56   −0.39 to0.34   −0.26 to 0.06 −0.37 to 0.42   −0.20 to −0.09 −0.62 to 0.49 Top7.21 to 8.88 5.38 to 6.95 3.79 to 5.13 −0.04 to 0.20 3.07 to 4.22 NA−45.66 to 65.37 LogEC50 −8.82 to −8.63 −9.00 to −8.74 −9.40 to −9.11−10.43 to −8.60 −9.56 to −9.23 NA −12.80 to −2.37 HillSlope 0.89 to 1.380.82 to 1.85 0.69 to 1.56 −3.47 to 6.61 0.64 to 1.90 NA −0.38 to 1.88NA—not applicable.MJ Cell Activation Assay Materials and Methods—Bead-BoundCoupling Proteins of Interest to Magnetic Particles

Anti-ICOS antibodies, control antibodies, and ICOSL-Fc, were coupled tobeads as follows.

Dynabeads M-450 Tosylactivated (Invitrogen; approximately 2×10^8beads/sample) were incubated with 100 μg of each protein sampleovernight at room temperature with agitation. Beads were washed threetimes with DPBS (Gibco) and incubated with 1M Tris-HCl, pH 8.0(UltraPure™, Gibco) for 1 hr at room temperature with agitation to blockthe uncoupled reactive sites. Beads were washed again three times withDPBS and finally resuspended in 0.5 ml of DPBS/sample.

The quantity of each protein of interest on the beads was thendetermined as follows. Black flat bottom, high binding ELISA plates(Greiner) were coated with Anti-human IgG (Southern Biotech) orAnti-Armenian Hamster IgG (Jackson ImmunoResearch) capture antibody at 4μg/ml in DPBS, 50 μl/well, overnight at 4° C. Wells were then washedthree times with DPBS+0.1% Tween, 200 μl/well and blocked with 200μl/well of DPBS+1% BSA for 1 hr at RT. Wells were washed again threetimes with DPBS+0.1% Tween. Stock protein samples were quantifiedspectrometrically and beads were counted on a cell counter. Dilutionseries of protein samples and beads were then incubated in the plates at50 μl/well for 1 hr at RT before washing again three times withDPBS+0.1% Tween. 50 μl/well of either biotinylated anti-armenian hamsterantibody or anti-human IgG—europium in DPBS+0.1% BSA were added andincubated for 1 hr at RT. In the case of addition of biotinylatedanti-Armenian hamster antibody C398.4A another incubation step with 50μl/well of streptavidin-europium (Perkin Elmer) diluted 1:500 in Assaybuffer (Perkin Elmer) were added and incubated for 1 hr at RT. The wellswere washed three times with 200 μl/well of TBS+0.1% Tween beforedeveloping the assay by adding 50 μl/well of Delfia enhancement solution(Perkin Elmer), incubating for 10 mins at RT and measuring thefluorescence emitted at 615 nm on the EnVision Multilabel Plate Reader.The quantity of protein on the beads was determined by extrapolatingvalues from the signals obtained from known concentrations of uncoupledprotein samples.

MJ Cell In Vitro Activation Assay—Bead Bound

MJ [G11] cell line (ATCC CRL-8294) was grown in IMDM (Gibco or ATCC)supplemented with 20% heat inactivated FBS. Cells were counted and 15000cells/well (50 μl/well) of cell suspension was added to 96-well clearflat bottom polystyrene sterile TC-treated microplates. Beads werecounted and serial 1:2 dilutions ranging from 1.5×10^6 beads/well toapproximately 5860 beads/well (50 μl/well) were added to the cells induplicate or in triplicate. To account for background several wells ofthe plate contained MJ cells only (100 μl/well). The cells and beadswere co-cultured in the plates for 3 days at 37° C. and 5% CO₂ afterwhich supernatants were harvested by centrifugation and collected forIFN-γ content determination.

Measuring IFN-γ Levels

The IFN-γ content in each well was determined using a modification ofthe Human IFNgamma DuoSet ELISA kit (R&D systems). Capture antibody (50μl/well) was coated overnight at 4 μg/ml in DPBS on black flat bottom,high binding plates (Greiner). The wells were washed three times with200 μl/well of DPBS+0.1% Tween. The wells were blocked with 200 μl/wellof 1% BSA in DPBS (w/v), washed three times with 200 μl/well ofDPBS+0.1% Tween and then 50 μl/well of either the IFN-γ standardsolutions in RPMI or neat cell supernatant were added to each well andincubated for 1 hr at RT. The wells were washed three times with 200μl/well of DPBS+0.1% Tween before adding 50 μl/well of biotinylateddetection antibody at 200 ng/ml in DPBS+0.1% BSA and incubated for 1 hrat RT. The wells were washed three times with 200 μl/well of DPBS+0.1%Tween before adding 50 μl/well of streptavidin-europium (Perkin Elmer)diluted 1:500 in Assay buffer (Perkin Elmer) and incubated for 1 hr atRT. The wells were washed three times with 200 μl/well of TBS+0.1% Tweenbefore developing the assay by adding 50 μl/well of Delfia enhancementsolution (Perkin Elmer) and incubating for 10 mins at RT and measuringthe fluorescence emitted at 615 nm on the EnVision Multilabel PlateReader.

Data Analysis

IFN-γ values for each well were interpolated from the standard curve andthe average background levels from cell-only wells were subtracted. Thebackground corrected values were then used in GraphPad prism to fit a4-parameter log-logistic concentration response curve.

Example 14: Monism of ICOS-Expressing MJ Cells by Plate-Bound Antibody

An alternative assay for agonism of ICOS-expressing T cells usesantibodies in a plate-bound format.

MJ Cell Activation Assay Materials and Methods—Plate-Bound

Antibody coating: 96-well, sterile, flat, high binding plates (Costar)were coated overnight at 4° C. with 100 μl/well of serial 1:2 dilutionsof proteins of interest (anti-ICOS antibodies, control antibodies, andICOSL-Fc) in DPBS (Gibco) ranging from 10 μg/ml to 0.02 μg/ml of induplicate or in triplicate. To account for background several wells ofthe plate were coated with DPBS only. Plates were then washed threetimes with 200 μl/well of DPBS before the addition of cells.

Cell stimulation: MJ [G11] cell line (ATCC CRL-8294) was grown in IMDM(Gibco or ATCC) supplemented with 20% heat inactivated FBS. The cellswere counted and 15000 cells/well (100 μl/well) of cell suspension wereadded to the protein coated plates. Cells were cultured in the platesfor 3 days at 37° C. and 5% CO₂. Cells were separated from the media bycentrifugation and the supernatants collected for IFN-γ contentdetermination.

Measurement of IFNγ levels and data analysis was as described in Example13.

Results

Results are shown in FIG. 13 and in Table E14-1 below. In summary,STIM001, STIM002 and STIM003 all showed potent agonism as measured byIFN-γ secretion with similar Log EC50 values (Log EC50 95% CI: −7.76 to−7.64, −7.79 to −7.70 and −7.82 to −7.73, respectively) and Top values(Top 95% CI: 2.06 to 2.54, 2.44 to 2.93 and 2.01 to 2.41, respectively).Clone C398.4A exhibited a similar Log EC50 value (Log EC50 95% CI: −7.78to −7.60) but lower Top value (Top 95% CI: 1.22 to 1.63) than STIM001 toSTIM003. STIM004 also showed agonism in this assay, but was less potent,reaching a moderate Top value (Top 95% CI: 0.16 to 0.82) with a similarLog EC50 value (Log EC50 95% CI: −7.91 to −7.21). STIM001, STIM002 andSTIM003 were stronger agonists than ICOSL-Fc (Log EC50 95% CI: −7.85 to−7.31 and Top 95% CI: 0.87 to 2.45).

TABLE E14-1 Summary of plate-bound MJ cell in vitro activation assay.Best-fit Clone values Clone C398.4A 95% CI STIM001 STIM002 STIM003STIM004 IgG1 C398.4A ICOSL-Fc IC Bottom −0.03 to 0.13   −0.08 to 0.11  −0.10 to 0.07   −0.06 to 0.07   NA −0.03 to 0.11   −0.16 to 0.09   −0.07to 0.04 Top 2.06 to 2.54 2.44 to 2.93 2.01 to 2.41 0.16 to 0.82 NA 1.22to 1.63 0.87 to 2.45   0.05 to 0.29 LogEC50 −7.76 to −7.64 −7.79 to−7.70 −7.82 to −7.73 −7.91 to −7.21 NA −7.78 to −7.60 −7.85 to −7.31 NAHillSlope 2.06 to 5.38  0.16 to 10.88 1.77 to 6.5  −1.46 to 6.77   NA1.24 to 8.20 0.26 to 3.97 NA IgG1 = Human IgG1 hybrid control antibody.

Example 15: Monism of ICOS-Expressing MJ Cells by Antibody in SolubleForm

In contrast to the assays described in Example 13 and Example 14, whichused antibody arrayed on a solid surface, this assay determines whetherantibody in soluble form acts as agonist of ICOS-expressing T cells.

MJ Cell Activation Assay Materials and Methods—Soluble

MJ [G11] cell line (ATCC CRL-8294) was grown in IMDM (Gibco or ATCC)supplemented with 20% heat inactivated FBS. Cells were counted and 15000cells/well (50 μl/well) of cell suspension was added to 96-well clearflat bottom polystyrene sterile TC-treated microplates. Serial 1:2dilutions of proteins of interest ranging from 10 μg/ml to 0.01953125μg/ml either alone or with the addition of a cross-linking reagent(AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, Fc Fragment Specific;Jackson ImmunoResearch) were added to the cells in duplicate or intriplicate (50 μl/well). To account for background several wells of theplate contained MJ cells only (100 μl/well). The cells and beads wereco-cultured in the plates for 3 days at 37° C. and 5% CO₂ after whichsupernatants were harvested by centrifugation and collected for IFN-γcontent determination.

Measurement of IFNγ levels and data analysis was as described in Example13.

Results

STIM001 and STIM002 both showed significant soluble agonism as measuredby IFN-γ secretion compared to Human IgG4.PE hybrid control. MAbcross-linking via Goat Anti-Human IgG Fc F(ab′)2 Fragment increasedsecreted IFN-γ levels even more.

Example 16: Binding of Antibody to Activated T Cells

A. Human ICOS

Ability of anti-ICOS antibodies to recognise the ICOS extracellulardomain in its native context on the surface of activated primary human Tcells is confirmed in this assay.

Pan T-cells (CD3 cells) were isolated and cultured for 3 days withCD3/CD28 dynabeads (Thermofisher) to induce ICOS expression on theirsurface. Surface staining of STIM001, STIM003 and the hIgG1 hybridcontrol (HC IgG1) was determined by two methods, namely detectionfollowing direct binding of pre-labelled antibodies (antibodies directlyconjugated with AF647) or indirectly via the use of a secondaryAF647-Goat anti-human Fc antibody. Stained cells were ran on the Attuneand staining intensity was presented as Mean of fluorescence intensity(MFI). EC50 was determined using GraphPad Prism.

Results are shown in FIG. 14. Once activated, pan CD3 T cells wereclearly stained by both STIM001 and STIM003 hIgG1. Notably, thesaturation of STIM003 binding to activated T cells occurred at a lowerconcentration than that of STIM001, suggesting higher affinity ofSTIM003 to human ICOS. The EC50 of STIM003 was roughly 100× lower thanthat of STIM001 (0.148 nM vs 17 nM for the indirect binding assay).

B. ICOS from Non-Human Primates

Ability of anti-ICOS antibodies to recognise the ICOS extracellulardomain in its native context on the surface of activated primary T cellsfrom non-human primates (NHP) is confirmed in this assay.

PBMC from whole blood of 2 Mauritian cynomolgus macaques (WickhamLaboratories) were isolated by gradient centrifugation and cultured for3 days with CD2/CD3/CD28 MACSiBeads (Miltenyi) to induce ICOS expressionon their surface. Surface staining of STIM001, STIM003 and the hIgG1hybrid control (HC IgG1) was determined following direct binding ofAF647 pre-labelled antibodies (from 80 μg to 8 pg/ml). Cells were alsolabelled with V450-CD3 to assess staining on T-cell subsets. Stainedcells were run on Attune (Thermofisher) and staining intensity waspresented as mean fluorescence intensity (MFI). EC50 was determinedusing GraphPad Prism.

Results are shown in FIG. 28. Once activated, T cells were clearlystained by both STIM001 and STIM003 hIgG1. As was observed with bindingto human T cells, saturation of STIM003 binding to activated NHP T cellsoccurred at lower concentration than that of STIM001, indicating thatSTIM0003 has the higher affinity of these two antibodies ICOS. EC50values for binding to NHP ICOS were similar to those obtained forbinding to human ICOS.

TABLE E16 EC50 (Molar) calculated for antibody binding to ICOS onactivated NHP T cells Pan T-cells EC50 Cynomolgus donor 1 Cynomolgusdonor 2 NHP STIM001 2.224e−7 not tested ST1M003 4.581e−9 4.830e−9 HumanSTIM001 2.209e−7 1.207e−7 ST1M003 2.293e−9 8.953e−10

Example 17: Analysis of T Cell Sub-Populations Among Tumour InfiltratingLymphocytes and Peripheral T Cells

A pharmacodynamics study revealed that anti-ICOS antibodies STIM001 andSTIM003 in mIgG2a isotype significantly deplete TRegs, increase thepercentage of CD4+ effector cells and increase the CD4+ effector/TRegratio as well as the CD8+/TReg ratio within the tumour microenvironment(TME).

The increased CD8+/TReg ratio and increased number of CD4+ effectorcells within the TME may collectively contribute to the CT26 tumourclearance observed when these anti-ICOS antibodies were co-injected withanti-PDL1 antibody in the STIM001 & STIM003 efficacy study (Example 11).

Method

The pharmacodynamics study was performed in female Balb/c mice bearingCT-26 mouse colon carcinoma cells (ATCC, CRL-2638). Balb/c mice weresupplied by Charles River UK at 6-8 weeks of age and >18 g and housedunder specific pathogen-free conditions. A total of 1×10E5 CT-26 tumourcells (passage number: P8) were subcutaneously injected in the rightflank. All CT-26 tumour bearing animals were assigned to 6 groups (TableE17-1) and individual mice were dosed twice (on Day 13 & Day 15 posttumour cell implantation) with 200 μg of antibody or saline. CD3+T-cells from the CT-26 tumour bearing animals were analysed by FACS onday 16 post tumour cell implantation.

TABLE E17-1 Treatment groups Number of Treatment regimen (Day 13 and Day15 post tumour Group animals cell implantation) 1 10 Saline 2 10Anti-ICOS (STIM001) mIgG1 200 μg each 3 10 Anti-ICOS (STIM001) mIgG2a200 μg each 4 10 Anti-ICOS (STIM003) mIgG1 200 μg each 5 10 Anti-ICOS(STIM003) mIgG2a 200 μg each 6 10 Anti-CTLA-4 (9H10) 200 μg eachResults

Animals treated with STIM001 & STIM003 in the mIgG2a isotype showed alower percentage of CD4+ CD3+ CD45+ cells at the tumour site whencompared with saline treated group (FIG. 15A), whereas STIM001 orSTIM003 treatments had very marginal effect of the percentage of CD8+CD3+ CD45+ cells at the tumour site (FIG. 15B). The decrease in CD4+ Tcells could be attributed to a profound decrease in the percentages ofT-Regulatory cells in all the groups treated with STIM001 and STIM003antibodies. Notably, animals treated with STIM001 and STIM003 in themIgG2a isotype showed a dramatic reduction in T-Regs (CD4+ Foxp3+ CD25+)within the TME, whereas STIM001 & STIM003 in the mIgG1 isotype had onlya modest effect on T-Reg content in TME. In addition, animals treatedwith STIM001 & STIM003 in the mIgG2a isotype had reduced T-Reg in theTME when compared with the animals treated with a commercial anti-CTLA-4(9H10, Biolegend Cat#106208) antibody which is known to deplete T-Reg[42], but this result did not reach statistical significance (FIG. 15C).The effect of STIM001 and STIM003 either in mIgG1 or mIgG2a isotypes onT-Reg compartment was more specific with tumour infiltrating lymphocytes(TILs). T-reg depletion was not observed in the periphery (as previouslydescribed for anti-CTLA4 [43]) (FIG. 15D). The changes in T-Reg contentsalso resulted in a significant increase in the percentage ofintra-tumoural CD4-effector cells (CD4+ Foxp3− CD25−) (FIG. 15E),similarly the ratio of CD4 effector/T-Reg and CD8/T-Reg ratio in theanimals treated with STIM001 & STIM003 in the mIgG2a was alsosignificantly increased within TME (FIG. 15F & FIG. 15G).

Example 18: Effect of Anti-ICOS Antibody on Level of ICOS-Expressing TCells in CT26 Tumour and Spleen

Analysis was performed to quantify the percentage of immune cells withinthe tumour compared with the spleen, by analysis of total immune cellsin the tumour and spleen tissues, following treatment with anti-ICOSantibody STIM001 or STIM003. STIM001 and STIM003 mIgG2a each caused asignificant reduction in Treg within the tumour, but not in the spleen,indicating a tumour-selective effect. This depletion was selective forTregs compared with other T cell subtypes. The results presented hereassist in understanding the effects of the STIM antibodies on the immunecontexture, and confirm that anti-ICOS antibodies witheffector-function-enabled Fc regions can strongly deplete TRegs.

Materials and Methods

Mice bearing CT26 tumours were dosed twice with STIM001, STIM003, oranti-CTLA4 antibody (9H10). The anti-CTLA4 antibody was included as apositive control for Treg depletion, as anti-CTLA4 antibody had beenpreviously shown to selectively reduce Tregs in tumours [43].

The immune contexture within the tumours and the spleen of treatedanimals was analysed by FACS following tissue disaggregation.

Details of FACS antibodies used in this study are shown in Table E18.All FACS antibodies were used at a concentration recommended by thesupplier. FACS data were acquired using Attune NxT flow cytometer anddata were analysed using FlowJo software.

TABLE E18 FACS antibodies. Marker Supplier Cat. number Lot numberFluorophore Live/dead Life L-34959 1784156 Fixable Yellow technologiesCD45 E-bioscience 45-0451-82 E08336-1636 PerCp-Cy5.5 CD3 E-bioscience48-0032-82 4278794 eFlour 450 CD4 E-bioscience 11-0042-86 E0084-1633FITC CD8 E-bioscience 12-0081-85 E01039-1635 PE Foxp3 E-bioscience17-5773-82 4291991 APC CD25 E-bioscience 47-0251-82 4277960 APC eF 780ICOS E-bioscience 25-9942-82 E17665-103 PE-CY7 Fc/Block E-bioscience16-0161-86 E06357-1633 —Results

ICOS expression was determined in the CT26 tumours and in the spleen oftumour-bearing animals. We observed an increased percentage of tumourinfiltrating immune cells expressing ICOS protein (FIG. 20), indicatingthat immune cells in the tumours are more often positive for ICOSexpression than immune cells in the periphery. TRegs in the tumour ofuntreated animals were nearly all (>90%) positive for ICOS expression,whereas CD8+ effector T cells in the tumour were not (approx. 60%).Comparing T cell subpopulations (again in untreated mice) in tumour withthose in spleen, a significantly higher (p<0.0001) percentage ofintratumoural Tregs were positive for ICOS compared with Tregs inspleen, and a significantly higher (p<0.001) percentage of intratumouralCD4+ Teff cells were positive for ICOS compared with CD4+ Teff cells inspleen.

Also in the mice before treatment, the level of ICOS expression was muchhigher on immune cells in the microenvironment of CT26 tumours, whencompared with immune cells in the spleen (FIG. 21). ICOS expression wasincreased on the surface of all immune cell subsets analysed (CD8T-Effector, CD4 T-Effector and CD4/FoxP3 TReg cells) in the tumourmicroenvironment. Note that although immune cells in the tumours and thespleen are both expressing ICOS, immune cells in the tumour areexpressing significantly more ICOS (indicated by higher MFI, FIG. 21)than cells in the spleen (indicated by lower MFI, FIG. 21). Importantly,TRegs in the tumour are expressing the highest levels of ICOS, aspreviously reported [11].

CT26 tumour bearing animals were treated with 2 doses of antibodySTIM001 or STIM003 and with an anti-CTLA-4 antibody. The STIM antibodiesdid not affect the overall percentage of CD45 positive cells (a markerfor immune cells) in the tumours, when used in either mIgG1 or mIgG2format. Nor did treatment with these antibodies significantly affect thepercentage of CD8 effector T cells in CT26 tumours (FIG. 22). Treatmentwith STIM001 in mIgG2a isotype led to a significant (p<0.05) depletionof CD4+ effector T cells, but none of STIM001 mIgG1, STIM003 mIgG1 andSTIM003 mIgG2a affected the percentage of CD4+ effector T cells.

Anti-CTLA-4 treatment produced a notable (albeit not statisticallysignificant) increase in CD45+ cells and CD8+ effector T cells in theTME, but did not affect CD4+ effector T cells (FIG. 22).

The STIM antibodies significantly affected regulatory T cells in thetumour. As shown in FIG. 23, STIM001 mIgG2a and STIM003 mIgG2asignificantly and selectively depleted TRegs (which are high for ICOSexpression) in the tumour microenvironment. Interestingly, theanti-CTLA4 antibody which, despite being included as a positive controlfor TReg depletion in this experiment, was less effective than the STIMmIgG2a antibodies at depleting TRegs.

This selective depletion of TRegs resulted in an increase in the ratioCD8 effector T cells to TRegs in the tumour, and an increase in theratio of CD4 effector T cells to TRegs in the tumour, both of whichshould favour an anti-tumour immune response. Ratio data are shown inFIG. 24.

In contrast to the depletion of intratumoural Tregs by STIM001 mIgG2aand STIM003 mIgG2a, no such effect was observed on Tregs in spleen (FIG.25, FIG. 26, FIG. 27). This indicates that the effects of the anti-ICOSantibodies on depletion of Tregs depletion was not systemic in all thetissues. Such selectivity could be advantageous for therapeuticanti-ICOS antibodies in treating tumours in patients, as preferentialdepletion of Tregs in the tumour microenvironment could selectivelyrelieve suppression of anti-tumour effector T cells, while minimisingside effects at other sites in the body. The anti-ICOS antibodies maythus promote an anti-tumour response in the immune system with a lowrisk of undesirable activation of a wider T cell response that couldcause treatment-limiting autoimmune adverse events.

Example 19: Antibody Stability

STIM003 human IgG1 was tested for stability during storage,freeze/thawing and purification, and was found to maintain its stabilityunder all tested conditions. % aggregation was determined by HPLC.

There was no significant change in the percentage of monomer (>99%)after 3 months storage at 4° C. in buffer (10 mM sodium phosphate, 40 mMsodium chloride, pH 7.0).

On thermal denaturation testing, all samples (n=15) had the same Tm (nosignificant difference between aliquots) and had comparable thermaldenaturation curves.

There was no significant change in Tm (≈70.3° C.), the percentage ofmonomer or the profile on SDS-PAGE after 3 cycles of freeze and thaw.

There was no significant change in Tm (≈70.3° C.), the percentage ofmonomer or the profile on SDS-PAGE after 7 days' storage at roomtemperature.

There was 90% recovery post protein A purification.

Example 20: Monotherapeutic Efficacy of Anti-ICOS Ab Against A20 TumourGrowth in Mouse

Anti-ICOS antibodies STIM001 mIgG2a and STIM003 mIgG2a each showedstrong anti-tumour efficacy when used as monotherapies in vivo in amouse A20 syngeneic model.

Materials and Methods

The efficacy study was performed in BALB/c mice using the sub-cutaneousA20 reticulum cell sarcoma model (ATCC, TIB-208). The A20 cell line is aBALB/c B cell lymphoma line derived from a spontaneous reticulum cellneoplasm found in an old BALB/cAnN mouse. This cell line has beenreported to be positive for ICOSL.

BALB/c mice were supplied by Charles River UK >18 gram and housed underspecific pathogen-free conditions. A total of 5×10e5 A20 cells (passagenumber below P20) were subcutaneously injected into the right flanks ofmice. The A20 cells were passaged in vitro washed twice in PBS andre-suspended in RPMI supplemented with 10% foetal calf serum. Cellviability was confirmed to be above 85% at the time of tumour cellinjection. Unless stated otherwise, antibody or isotype administrationwas initiated from day 8 post tumour cells injection.

STIM001 and STIM003 anti-ICOS antibodies were generated in mouse IgG2aisotype format. The mouse cross reactive anti-PD-L1 antibody (AbW) wasalso generated in the same isotype format (mouse IgG2a). STIM001,STIM003 and anti-PD-L1 antibodies were dosed intraperitoneally (IP) at200 μg of each antibody twice a week starting from day 8 (dosing for 3weeks between day 8-29) post tumour cell implantation. Animal weightsand tumour volume were measured 3 times a week from the day of tumourcell injection. Tumour volume was calculated by use of the modifiedellipsoid formula 1/2(Length×Width2). Mice were kept on study untiltheir tumour reached an average diameter of 12 mm. The experiment wasstopped at day 43 post tumour cell implantation. Tumour growth wasmonitored and compared with tumours of animals treated with isotypecontrol (mIgG2a) antibody. Treatment groups are shown in Table E20below.

TABLE E20 Treatment groups for A20 study. Number of Treatment regimenGroup animals (twice per week for 3 weeks 7 doses) 1 8 mIgG2a isotypecontrol 200 μg/mouse/each dose 2 8 Anti-PD-L1 mIGg2a (AbW) 200μg/mouse/each dose 3 8 Anti-ICOS mIgG2a STIM001 200 μg/mouse each dose 48 Anti-ICOS mIgG2a STIM003 200 μg/mouse/each doseResults

Monotherapy administration of either STIM001 or STIM003 (mIgG2a) in theA20 tumour model produced a complete anti-tumour response (FIG. 32, FIG.33). All the animals administered with either STIM001 or STIM003 werecured of the disease. This contrasts with the results in the isotypecontrol and PD-L1 mIgG2a groups (FIG. 30, FIG. 31). In rare cases,regression of tumours was observed for some animals in the isotypecontrol (spontaneous regression) and anti-PDL-1 groups, but treatmentwith anti-ICOS antibody produced significantly greater efficacy. At theend of the study, 3 of 8 control animals and 2 of 8 anti-PDL-1 treatedanimals had no tumour. However, all animals treated with either STIM001or STIM003 were tumour free at the end of the study (8 of 8 mice in bothgroups), representing 100% cure using the anti-ICOS antibodies.

Example 21: Strong Anti-Tumour Efficacy In Vivo in the J558 MyelomaSyngeneic Model for Combination of Anti-ICOS Antibody and Anti-PD-L1Antibody

Anti-ICOS antibody STIM003 mIgG2a and anti-PD-L1 antibody AbW mIgG2awere administered individually and in combination in the J558 tumourmodel. This is a syngeneic mouse model of myeloma. The anti-ICOSantibody was found to inhibit tumour growth when dosed as monotherapy orin combination with anti-PD-L1.

Materials & Methods

Anti-tumour efficacy studies were performed in Balb/c mice using thesub-cutaneous J558 plasmacytoma:myeloma cell line (ATCC, TIB-6). Balb/cmice were supplied by Charles River UK at 6-8 weeks of age and >18 g andhoused under specific pathogen-free conditions. A total of 5×10⁶ cells(passage number below P15) were subcutaneously injected (in 100 μl) intothe right flanks of mice. Unless stated otherwise, on day 11 post tumourcells injection, the animals were randomised based on tumour size andtreatments were initiated. The J558 cells were passaged in vitro byusing TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS andresuspended in DMEM supplemented with 10% foetal calf serum. Cellviability was confirmed to be above 90% at the time of tumour cellinjection.

Treatment was initiated when the tumours reached an average volume of˜140 mm^3. Animals were then allocated to 4 groups with similar averagetumour size (see Table E-21 for the dosing groups). Both antibodies,which are mouse cross-reactive, were dosed IP from day 11 (post tumourcell implantation) twice a week for 3 weeks (FIG. 38) unless the animalshad to be removed from study due to welfare (rare) or tumour size. As acontrol, a group of animals (n=10) was dosed at the same time using asaline solution. For the combination group, both STIM003 and anti-PDL1antibodies were dosed concurrently IP at 60 μg and 200 μg respectively(in 0.9% saline). Tumour growth was monitored over 37 days and comparedto tumours of animals treated with saline. Animal weight and tumourvolume were measured 3 time a week from the day of tumour cellinjection. Tumour volume was calculated by use of the modified ellipsoidformula 1/2(Length×Width²). Mice were kept on studies until their tumourreached an average diameter of 12 mm³ or, in rare cases, when incidenceof tumour ulceration was observed (welfare).

TABLE E21 Treatment groups for J558 efficacy study. Number Groups ofanimals Treatment regimen twice per week from day 11 1 10 Saline 2 8Anti-PD-L1 mIgG2a 200 μg (AbW) 3 8 Anti-ICOS STIM003 mIgG2a/anti-PD-L1mIgG2a (AbW) combination 60 μg/200 μg (respectively) 4 8 Anti-ICOSSTIM003 mIgG2a 60 μgResults

J558 syngeneic tumours were highly aggressive and all the animals in thesaline control group (n=10) had to be removed from studies by day 21 dueto tumour size. The anti-STIM003 mIgG2a and the anti-PDL1 mIgG2a bothdemonstrated good efficacy as monotherapies in this model with 37.5% and75% of the animals cured of disease, respectively. Importantly,combination of the two antibodies resulted in 100% of the animals havingrejected the plasmacytoma tumours by day 37. Data are shown in FIG. 38.

Example 22: Administration of Anti-PD1 Increases ICOS Expression on TILsSignificantly More than Anti-PD-L1 Antibody

A pharmacodynamic study was performed in animals harbouring establishedCT26 tumours to evaluate the effect of treatment with anti-PD-L1 oranti-PD-1 antibodies on ICOS expression on subsets of tumourinfiltrating lymphocytes (TILs). The following antibodies were compared:

-   -   anti-PD-L1 AbW mIgG1 [limited effector function]    -   anti-PD-L1 AbW mIgG2a [with effector function]    -   anti-PD-L1 10F9.G2 rat IgG2b [with effector function]    -   anti-PD1 antibody RMT1-14 rat IgG2a [effector null].

Tumours of treated mice were isolated, dissociated to single cells andstained for CD45, CD3, CD4, CD8, FOXP3 and ICOS.

Materials & Methods

Rat anti-PD-1 RMP1-14 IgG2a (BioXCell; Catalog number: BE0146), ratanti-PD-L1 10F9.G2 IgG2b (Bio-Legend; Catalog number: 124325) andanti-PD-L1 AbW mIgG1 and mIgG2a were tested in the CT26 tumour model bydosing i.p. with 130 μg on days 13 and 15 post tumour cell implantation.On day 16, animals were culled and the mouse tumours were harvested forFACS analysis. Tumours were dissociated using a mouse tumourdissociation kit (Miltenyi Biotec) and homogenised. The resulting cellsuspensions were clarified through 70 μM filters, pelleted andresuspended in FACS buffer at 2 million cells/well in a 96 well plate.The cell suspensions were incubated with anti-16/32 mAb (eBioscience)and stained with FACS antibodies specific for CD3 (17A2), CD45 (30-F11),CD4 (RM4-5), CD8 (53-6.7) and ICOS (7E.17G9) all obtained fromeBioscience Ltd. Cells were also stained with LiveDead Yellow fixableviability dye (Life technologies). For the Foxp3 intracellular staining,samples were fixed, permeabilised, and stained with antibody specificfor Foxp3 (eBioscience, FJK-16s). The samples were resuspended in PBSand data aquired on the Attune flow cytometer (Invitrogen) and analysedusing FlowJo V10 software (Treestar).

Results

Treatment with anti-PD1 and anti-PD-L1 antibodies only resulted in amarginal increase in the percentage on CD8 cells and T Regs expressingICOS at the measured timepoint. However, in response to anti-PD1 ratIgG2a, a clear and significant (over the saline treated group) increasein ICOS expression (increased dMFI) was observed on the surface ofICOS+ve CD8 cells. ICOS expression was also noted to be upregulated onCD4 effector and CD4 T Reg cells although this did not reach statisticalsignificance. This anti-PD1 antibody induced a marked increase in ICOSexpression on CD8 effector cells that was barely seen with theanti-PD-L1 mIgG2a. Similarly, when comparing the different formats ofanti-PD-L1 antibodies, in some of the animals treated it was observedthat the antibody having the lowest effector function (mIgG1) wasassociated with higher ICOS expression on effector CD8 and CD4 cellswhen compared with antibody having effector function (mIgG2a andratIgG2b), which rarely showed this. See FIG. 39.

An increase in ICOS expression on effector CD8/CD4 T cells may have theeffect of rendering these cells more sensitive to depletion by anti-ICOSantibody (e.g., on treatment of mice with STIM003 mIgG2a). An antibodythat exhibits lower ICOS induction in effector CD8 and CD4 T cells maybe preferable for use in combination with anti-ICOS antibody. The datafrom this study indicate that anti-PD-L1 effector positive antibody maybe especially suitable for combination with anti-ICOS effector positiveantibody, reflecting the anti-tumour efficacy observed when combininganti-PDL1 mIgG2a with STIM003 mIgG2a reported in other Examples herein.

Example 23: Strong Anti-Tumour Efficacy of Single Dose Anti-ICOSAntibody Monotherapy In Vivo in a B Cell Lymphoma Syngeneic Model

This experiment confirms the anti-tumour efficacy of STIM003 mIgG2a asmonotherapy. Strong anti-tumour efficacy was demonstrated after shortexposure of STIM003 mIgG2a.

Materials & Methods

Efficacy studies were performed in BALB/c mice using the sub-cutaneousA20 Reticulum Cell Sarcoma model (ATCC number CRL-TIB-208). BALB/c micewere supplied by Charles River UK at 6-8 weeks of age and >18 g andhoused under specific pathogen-free conditions. A total of 5×10E5 A20cells (passage number below P20) were subcutaneously injected into theright flanks of mice. Treatments were initiated at day 8 post tumourcells injection as shown in the table below. The A20 cells were passagedin vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice inPBS and resuspended in RPMI supplemented with 10% foetal calf serum.Cell viability was confirmed to be above 85% at the time of tumour cellinjection. STIM003 mIgG2a was used either as a single dose (SD) of 60 μg(equivalent to 3 mg/kg for a 20 g animal) or as multiple doses (MD,twice a week for 3 weeks) of 60 μg. Anti-tumour efficacy observed inresponse to the two schedules was compared to that of animals “treated”with saline (MD, twice a week for 3 weeks). The antibodies were dosedintraperitoneal (IP) as 1 mg/ml in 0.9% saline. Animal weight and tumourvolume were measured 3 times a week from the day of tumour cellinjection. Tumour volume was calculated by use of the modified ellipsoidformula 1/2(Length×Width²). Mice were kept on study until their tumourreached an average diameter of 12 mm or, rarely, when incidence oftumour ulceration was observed (welfare).

TABLE E23-1 Treatment groups. Number of Group animals Treatment regimen(IP injection) 1 10 Saline (multiple dose from day 8, twice a week for 3weeks) 2 10 STIM003 mIgG2 A (multiple dose from day 8, twice a week for3 weeks) 3 10 STIM003 mIgG2 A (Single dose on day 8)Results

Both multiple and single dose of STIM003 mIgG2a resulted in strong andsignificant monotherapy anti-tumour efficacy as shown by the number ofanimals with no signs of tumour growth at endpoint (Day 41). SD resultedin 7 our 10 animals cured from the disease whereas the multiple dosecured 9 out of 10 animals injected with A20 B cell lymphoblast. Allanimals in the saline treated group had to be removed from the study byday 40 due to tumour size. See FIG. 40.

Humane endpoint survival statistics were calculated from theKaplan-Meier curves (FIG. 41) using GraphPad Prism V7.0. This approachwas used to determine if the treatments were associated with improvedsurvival. The Hazard Ratio (Mantel-Haenszel) values and their associatedP values (Log-Rank Mantel-Cox) are shown in the table below.

TABLE E23-1 Hazard Ratio (Mantel-Haenszel) values and their associated Pvalues (Log-Rank Mantel-Cox) corresponding to FIG. 41 Kaplan-Meiercurves. Hazard Ratio (Mantel- Haenszel) MD vs Saline SD vs Saline MD vsSD Ratio (and its 0.09995 0.1076 0.5314 reciprocal) 95% CI of ratio0.02604 to 0.3837 0.02856 to 0.4052 0.05522 to 5.115 P Value 0.00080.001 0.5842

Example 24: Time and Dose Dependent Effects of Anti-ICOS Antibody inCT-26 Tumour Bearing Animals

This Example presents the results of a pharmacodynamic study evaluatingthe effects of anti-ICOS antibody on immune cells in mice bearing CT-26tumours. T and B cell subtypes from different tissues were analysed byFACS after a single dose of STIM003 mIgG2a.

Methods

CT-26 tumour bearing animals were dosed i.p. with either saline orSTIM003 at 200 μg, 60 μg or 6 μg on day 12 post tumour cellimplantation. Tumour tissues, blood, tumour draining lymph node (TDLN)and spleen were harvested on day 1, 2, 3, 4, and day 8 post treatment.The tumours were dissociated to make single cell suspension using mousetumour dissociation kit (Miltenyi Biotec). Spleen tissue was dissociatedusing gentle MACS dissociation, red blood cells were lysed using RBClysis buffer. Tumour draining lymph nodes were mechanicallydisaggregated to make single cells suspensions. The resulting cellsuspensions were clarified through either 70 μM or 40 μM filtersdepending on the tissue, cells were then washed twice in RMPI completemedia and finally resuspended in ice cold FACS buffer. Total blood wascollected into plasma tubes and red blood cells were lysed using RBClysis buffer, cells were washed twice in RMPI complete media and finallyresuspended in ice cold FACS buffer. The single cell suspension from allthe tissues were distributed into 96 deep well plates for FACS analysis.Cells were stained with Live Dead Fixable Yellow viability dye (Lifetechnologies). The cell suspensions were incubated with anti-CD16/CD32mAb (eBioscience) and stained with FACS antibodies specific for CD3(17A2), CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61.5), ICOSL(HK5.3), B220 (RA3-6B2), Ki-67 (SolA15), CD107a (eBio1D4B), IFN-γ(XMG1.2), TNF-α (MP6-XT22), Foxp3 (FJK-16s) and ICOS (7E.17G9) allobtained from eBioscience Ltd. For cytokine readout by FACS, singlecells suspensions from the tumours were plated in 24 well plate for 4hours in the presence of Brefeldin-A. For the intracellular staining,samples were fixed, permeabilised, and stained with specific antibodies.The samples were finally resuspended in PBS and data acquired on theAttune flow cytometer (Invitrogen) and analysed using FlowJo V10software (Treestar).

Results are presented and discussed below.

ICOS Expression is High on Intra-Tumoral T-Regs in the CT26 Model

When the percentage of tumour infiltrating lymphocytes (TILs) expressingICOS was compared to the percentage of immune cells in the spleen,blood, and TDLN, we demonstrated that more immune cells in themicroenvironment of CT-26 tumours expressed ICOS vs other tissues. Moreimportantly, the percentage of ICOS positive T-reg cells in all thetissues and at all the time points was higher than the percentage of CD4or CD8 effector T cells positive for ICOS. Importantly, the dMFI(relative expression) for ICOS also followed the similar ranking inexpression with intra-tumoural T-reg being highly positive for ICOSexpression vs other TILs subtypes. Interestingly, there was no strikingchange in the percentage of ICOS⁺ TILs within the time frame of thisexperiment. Similar results were also seen in spleen and TDLN. On theother hand, in the blood, ICOS expression is relatively stable on Teffector cells but increased on T-regs during the course of theexperiment. Altogether the data demonstrated that more cells expressedICOS in the tumour microenvironment and these positive cells alsoexpressed more ICOS molecules on their surface. More importantly, T regsin TILs are highly positive for ICOS. See FIG. 42.

Strong Depletion of Intra-Tumoural T-Reg Cells in Response to STIM003Administration

In response to the STIM003 mIgG2a antibody, there was strong and rapiddepletion of T-reg cells (CD4+CD25+Foxp3) in TME. As T-regs have highICOS expression compared with the other T cells subsets, it is expectedthat an anti-ICOS antibody with effector function would preferentiallydeplete these cells. At the lower dose of STIM003 (6 μg corresponding toa 0.3 mg/kg for a 20 g animal) there was a continuous depletion of T-regand by day 3 most of the T-reg were depleted from TME. Interestingly, byday 8, T-reg cells repopulate the TME then reach a level slightly abovethat observed in the saline treated animals. The repopulation of T-regcells at lower dose can be attributed to the increase in theproliferating CD4 T cells in TME as evidenced by an observed increase inKi-67+ CD4 T-cells. At a dose higher than 6 μg there was a long-termdepletion of T-reg cells in TME as shown by full T Reg depletion untilthe last time point analysed in this study (day 8). Whereas in the bloodthere was a transient depletion of T-reg cells at all doses.Importantly, by day 8, all the treated animals had similar (or higherfor the 6 μg dose) level of T-reg cells in the blood when compared tothe saline treated animals. Data are shown in FIG. 43. Notably, andsimilarly to data previously published for depleting CTLA-4 antibodies,there was no significant change in the percentage of T-reg cells in thespleen or TDLN tissues, suggesting that T-reg cells may be protectedfrom depletion in these organs.

In summary, strong depletion of T-reg cells in TME was achieved in CT-26model at a dose as low as 6 μg per animal. However, a dose of 60 μgresulted in long term depletion up to 8 days post STIM003 mIgG2ainjection. This was not improved by using higher dose (200 μg).

STIM003 mIgG2a Increased CD8:T Reg and CD4:T Reg Ratios

Effects of STIM003 on T-eff:T-reg ratios are shown in FIG. 44.

STIM003 mIgG2a increased the CD8:T-reg ratio as well as the CD4eff:T-reg ratio. Although all the treatment doses were associated withan increase in T-eff to T-reg ratio, the intermediate dose of 60 μg (theequivalent of 3 mg/kg for a 20 g animals) was associated with thehighest ratio by day 8 post treatment.

Interestingly, at the 6 μg dose, the ratios were high until day 4 but byday 8 post treatment they were matching that of the saline treatedanimals. This can be explained by the repopulation of TRegs observed forthis dose by day 8 post treatment. On the other hand, at a dose of 60 or200 μg, the Teff to T-reg ratios remained high at all time points. Thisis explained by a long term depeletion of Tregs at these doses. Notably,at higher dose (200 μg), despite the long term Treg depletion there wasonly a moderate improvement in the ratio by day 8. This can be explainedby some depletion of ICOS^(INT) effector cells at high concentration ofSTIM003.

Altogether, the data demonstrated TReg depletion and increasedEffector:T reg ratio at all doses tested. However, an optimal dose of 60μg (˜3 mg/kg) achieved both a long-term depletion of T-reg, as well asthe highest T-eff to T-reg ratios which would be associated with themost favourable immune context to initiate an anti-tumour immuneresponse. Interestingly a similar pattern was observed in the blood,with the intermediate dose of 60 μg associated with the highest T-eff toT-reg ratio. Importantly, in the blood, improvement of the ratio wasobserved at an earlier time point (between day 3 and day 4).

Activation of Effector Cells in Response to STIM003

Surface expression of CD107a on the tumour infiltrating T effector cellswas previously identified as a reliable marker for cells that have beenactivated and exert cytotoxic activity [44]. In the present studyemployed this marker to confirm that STIM003, in addition to depletingT-regs, can stimulate the cytotoxic activity of effector T cells in theTME. Interestingly, on day 8 post treatment, there was an increase insurface expression of CD107a on both the CD4 and CD8 effector T cellcompartments at all doses of STIM003. Furthermore, this upregulation ofCD107a expression on the surface on both CD4 and CD8 T cells appeared toplateau when animals were dosed at 60 μg as no improvement was seen at200 μg dosing.

To further demonstrate activation of effector cells in the TME, thecytokine release by CD4 and CD8 TILs was analysed by FACS. As expectedand consistent with the in-vitro agonism data presented in earlierExamples herein, STIM003 mIgG2a at all doses promoted pro-inflammatorycytokine IFN-γ and TNF-α production by effector CD4 and CD8 T cells. Theinduction of pro-inflammatory cytokine production appeared to be high atthe optimum dose of 60 μg. Indeed, 60 μg of STIM003 significantlyincreased cytokine production by CD4 T cells. A similar trend was seenfor the proinflammatory cytokine IFN-γ and TNF-α production by effectorCD8 T cells in TME. Data are shown in FIG. 45.

In summary, STIM003 at all the doses resulted in T cells activation inthe TME as shown by (1) the presence of the degranulation marker CD107aon their surface and (2) by the production of Th1 cytokines (IFNγ andTNFα) by T cells. This indicates that STIM003 strongly affects theimmune context in the TME and plays the dual role of depleting Tregcells and stimulate the killing activity of T effector cells.

Human Dose Estimations

Based on the pre-clinical efficacy data seen in mice, initialpredictions can be made of the clinical dose appropriate for humanpatients, based on corresponding biological surface area (BSA) [45].

For example, taking the optimal anti-ICOS IgG dose in mouse to be 3mg/kg (60 μg), and following the methods of ref. [45], the correspondingdose for a human is 0.25 mg/kg.

Using the Mosteller formulae, for an individual of 60 kg and 1.70 m theBSA 1.68 m². Multiplying the dose in mg/kg by a factor of 35.7 (60/1.68)gives a fixed dose of 15 mg. For an individual of 80 kg thecorresponding fixed dose would be 20 mg.

Doses may be optimised for human therapy in clinical trials to determinesafe and effective treatment regimens.

Example 25: Bioinformatic Analysis of Data from Tumour Samples

One target group of cancers according to the present invention is thosecancers that are associated with a relatively high level of ICOS+immunosuppressive Tregs.

To identify cancer types associated with a high content of Tregs,transcriptome data was obtained from The Cancer Genome Atlas (TCGA)public dataset and analysed for ICOS and FOXP3 expression levels. TCGAis a large-scale study that has catalogued genomic and transcriptomicdata accumulated for many different types of cancers, and includesmutations, copy number variation, mRNA and miRNA gene expression, andDNA methylation along with substantial sample metadata.

Gene Set enrichment analysis (GSEA) was conducted as follows. Geneexpression RNA seq data collected as part of the TCGA consortium wasdownloaded from the UCSC Xena Functional Genomics Browser as log2(normalized_count+1). Non-tumour tissue samples were removed from thedataset, leaving data for 20530 genes from 9732 samples. An algorithmfrom [46] and its implementation in [47] that calculates enrichmentscores for genes within a specified gene set was used to transpose genelevel counts to gene set scores for each sample. The gene set ofinterest was defined as containing both ICOS and FOXP3. Samples weregrouped by primary disease and the ssGSEA scores for each group werecompared across the 33 primary disease groups. The disease groups thatshowed the highest median scores were found to be lymphoid neoplasmdiffuse large b-cell lymphoma, thymoma, head and neck squamous cellcarcinoma, although diffuse large b-cell lymphoma showed a multimodaldistribution of scores with a subset scoring highly and the rest scoringbelow the group median.

In rank order of highest to lowest ssGSEA score for ICOS and FOXP3expression, the top 15 cancer types were:

DLBC (n = 48) lymphoid neoplasm diffuse large b-cell lymphoma THYM (n =120) thymoma HNSC (n = 522) head and neck squamous cell carcinoma TGCT(n = 156) testicular germ cell tumour STAD (n = 415) stomachadenocarcinoma SKCM (n = 473) skin cutaneous melanoma CESC (n = 305)cervical squamous cell carcinoma and endocervical adenocarcinoma LUAD (n= 517) lung adenocarcinoma LAML (n = 173) acute myeloid leukemia ESCA (n= 185) esophageal carcinoma LUSC (n = 502) lung squamous cell carcinomaREAD (n = 95) rectum adenocarcinoma COAD (n = 288) colon adenocarcinomaBRCA (n = 1104) breast invasive carcinoma LIHC (n = 373) liverhepatocellular carcinoma

In which n is the number of patient samples for that cancer type in TCGAdataset. Anti-ICOS antibodies described herein may be used for treatmentof these and other cancers.

Cancers that are associated with a relatively high level of ICOS+immunosuppressive Tregs and which further express PD-L1 may respondespecially well to treatment with a combination of anti-ICOS antibodyand anti-PD-L1 antibody. Appropriate treatment regiments and antibodiesfor this purpose have already been detailed in the foregoingdescription.

Using the TCGA dataset as before, enrichment scores for ICOS and FOXP3were correlated with expression levels of PD-L1 using Spearman's rankcorrelation and grouped by primary disease indication. P-values werecalculated for each group and a p-value of 0.05 (with Bonferroni'smultiple comparison correction) was taken as statistically significant.The disease groups with the highest correlations between ICOS/FOXP3 andPD-L1 expression were:

TGCT (n = 156) testicular germ cell tumour COAD (n = 288) colonadenocarcinoma READ (n = 95) rectum adenocarcinoma BLCA (n = 407)bladder urothelial carcinoma OV (n = 308) ovarian serouscystadenocarcinoma BRCA (n = 1104) breast invasive carcinoma SKCM (n =473) skin cutaneous melanoma CESC (n = 305) cervical squamous cellcarcinoma and endocervical adenocarcinoma STAD (n = 415) stomachadenocarcinoma LUAD (n = 517) lung adenocarcinoma

Patients may be selected for treatment following an assay determiningthat their cancer is associated with ICOS+ immunosuppressive Tregs andexpression of PD-L1. For cancer types in which, as above, there is ahigh correlation score, it may suffice to determine that one of ICOS+immunosuppressive Tregs and expression of PD-L1 is present (e.g., abovea threshold value). PD-L1 immunohistochemistry assays may be used inthis context.

Example 26: Assessment of Further Anti-ICOS Antibodies

CL-74570 and CL-61091 antibody sequences identified in Example 12 weresynthesised and expressed in IgG1 format in HEK cells.

Functional characterisation of these antibodies was performed using anHTRF assay similar to that described in Example 6, with modifications toadapt the assay to use of purified IgG1 rather than BCT supernatant. 5μL of supernatant containing human IgG1 antibodies expressed from HEKcells was used in place of the BCT supernatant, and the total volumemade up to 20 μl per well using HTRF buffer as before. A human IgG1antibody was used as a negative control. Both antibodies exhibitedgreater than 5% effect for binding to human and mouse ICOS as calculatedusing Equation 1 and were therefore confirmed to test positive in thisassay.

Ability of these antibodies to bind human and mouse ICOS expressed onthe surface of CHO-S cells was further confirmed using a Mirrorballassay. In this assay, 5 μl supernatant containing the anti-ICOS IgG1 wastransferred to each well of 384 mirrorball black plates (Corning).Binding of anti-ICOS antibodies was detected by adding 10 μl of goatanti-human 488 (Jackson Immunoresearch) diluted in assay buffer (PBS+1%BSA+0.1% Sodium Azide) at a concentration of 0.8 mg/ml to all wells.

For positive control wells, 5 μL reference antibody diluted in assaymedia to 2.2 μg/mL was added to the plates. For negative control wells,5 μl of Hybrid control IgG1 diluted in assay media to 2.2 μg/mL wasadded to the plates. 10 μM of DRAQ5 (Thermoscientific) was added to0.4×10⁶/ml cells resuspended in assay buffer and 5 μl was added to allwells. Plates were incubated for 2 hr at 4 degrees.

Fluorescence intensity was measured using Mirrorball plate reader (TTPLabtech), measuring Alexafluor 488 (excitation 493 nm, emission 519 nm)from a population of 500-700 single cells. Assay signal was measured asMedian (FL2) Mean Intensity.

Total binding was defined using reference antibody at an assayconcentration of 2.2 μg/mL. Non-specific binding was defined usingHybrid control hIgG1 at an assay concentration of 2.22 μg/mL. Bothantibodies exhibited greater than 1 percent effect and were thereforeconfirmed to test positive in this assay.

${{Percent}\mspace{14mu}{effect}} = {\frac{\left( {{{sample}\mspace{14mu}{well}} - {{non}\text{-}{specific}\mspace{14mu}{binding}}} \right)}{\left( {{{total}\mspace{14mu}{binding}} - {{non}\text{-}{specific}\mspace{14mu}{binding}}} \right)} \times 100}$

Each of CL-74570 and CL-61091 also demonstrated binding to human andmouse ICOS expressed on CHO-S cells as determined by flow cytometry.FACS screening was performed using a method similar to that described inExample 6, with modifications to adapt the assay to use of purified IgG1rather than BCT supernatant. Both antibodies exhibited binding >10 foldabove the average of geomean of the negative control binding to hICOS,mICOS and WT CHO cells.

TABLE E26-1 Functional characterisation of CL-74570 and CL-61091.Primary Screen Mirrorball Secondary screen HTRF (Protein) (ICOS CHOCell) FACS Human Mouse Human Mouse Human ICOS CHO Mouse ICOS CHO 1:100dil 1:100 dil 1:100 dil 1:100 dil (1:10 dil) (1:10 dil) Percent PercentPercent Percent % % Clone Effect [%] Effect [%] Effect [%] Effect [%]Binding-APC Binding-APC ID 94.42 60.86 107.02 127.03 122.97 96.41CL-74570 83.43 76.65 54.14 113.10 19.08 62.94 CL-61091

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Sequences Antibody STIM001 VH domain nucleotide sequence: SEQ ID NO: 367CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 366QVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAVYYCARSSGHYYYYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GYTFSTFG SEQ ID NO: 363VH CDR2 amino acid sequence: ISAYNGDT SEQ ID NO: 364VH CDR3 amino acid sequence: ARSSGHYYYYGMDV SEQ ID NO: 365VL domain nucleotide sequence: SEQ ID NO: 374GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 373DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYCMQSLQTPLTFGGGTKVEIKVL CDR1 amino acid sequence: QSLLHSNEYNY SEQ ID NO: 370VL CDR2 amino acid sequence: LGS SEQ ID NO: 371VL CDR3 amino acid sequence: MQSLQTPLT SEQ ID NO: 372 Antibody STIM002VH domain nucleotide sequence: SEQ ID NO: 381CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 380QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO: 377VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO: 378VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO: 379VL domain nucleotide sequence: 388GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACorrected STIM002 VL domain nucleotide sequence: SEQ ID NO: 519GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 387DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLSFGQGTKLEIKVL CDR1 amino acid sequence: QSLLHSDGYNY SEQ ID NO: 384VL CDR2 amino acid sequence: IGS SEQ ID NO: 385VL CDR3 amino acid sequence: MQALQTPLS SEQ ID NO: 386 Antibody STIM002-BVH domain nucleotide sequence: SEQ ID NO: 395CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 394QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO: 391VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO: 392VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO: 393VL domain nucleotide sequence: SEQ ID NO: 402GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 401DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPCSFGQGTKLEIKVL CDR1 amino acid sequence: QSLLHSDGYNC SEQ ID NO: 398VL CDR2 amino acid sequence: IGS SEQ ID NO: 399VL CDR3 amino acid sequence: MQALQTPCS SEQ ID NO: 400 Antibody STIM003VH domain nucleotide sequence: SEQ ID NO: 409GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCACorrected STIM003 VH domain nucleotide sequence: SEQ ID NO: 521GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 408EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDFYGSGSYYHVPFDYWGQGILVTVSSVH CDR1 amino acid sequence: GVTFDDYG SEQ ID NO: 405VH CDR2 amino acid sequence: INWNGGDT SEQ ID NO: 406VH CDR3 amino acid sequence: ARDFYGSGSYYHVPFDY SEQ ID NO: 407VL domain nucleotide sequence: SEQ ID NO: 416GAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA VL domain amino acid sequence: SEQ ID NO: 415EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYDMSPFTFGPGTKVDIKVL CDR1 amino acid sequence: QSVSRSY SEQ ID NO: 412VL CDR2 amino acid sequence: GAS SEQ ID NO: 413VL CDR3 amino acid sequence: HQYDMSPFT SEQ ID NO: 414 Antibody STIM004VH domain nucleotide sequence:GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 423VH domain amino acid sequence:EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEWVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDYYGSGSYYNVPFDYWGQGTLVTVSS SEQ ID NO: 422VH CDR1 amino acid sequence: GLTFDDYG SEQ ID NO: 419VH CDR2 amino acid sequence: INWNGDNT SEQ ID NO: 420VH CDR3 amino acid sequence: ARDYYGSGSYYNVPFDY SEQ ID NO: 421VL domain nucleotide sequence: SEQ ID NO: 431GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAAVL domain amino acid sequence as encoded by the above VL domain nucleotidesequence. Corrected VL domain nucleotide sequence: SEQ ID NO: 430GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAA Corrected VL domain amino acid sequence: SEQ ID NO: 432EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYGSSPFFGPGTKVDIKVL CDR1 amino acid sequence: QSVSSSY SEQ ID NO: 426VL CDR2 amino acid sequence: GAS SEQ ID NO: 427VL CDR3 amino acid sequence: QQYGSSPF SEQ ID NO: 428 Antibody STIM005VH domain nucleotide sequence: SEQ ID NO: 439CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCAVH domain amino acid sequence: SEQ ID NO: 438QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGI IWVRQAPGQGLEWMGWI SVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAVYYCARAGYDILTDFSDAFDIWGHGTMVTVSSVH CDR1 amino acid sequence: GYTFNSYG SEQ ID NO: 435VH CDR2 amino acid sequence: ISVHNGNT SEQ ID NO: 436VH CDR3 amino acid sequence: ARAGYDILTDFSDAFDI SEQ ID NO: 437VL domain nucleotide sequence: SEQ ID NO: 446GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAACTT TTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAA VL domain amino acid sequence: SEQ ID NO: 445DIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYGIPWVGQGTKVEIKVL CDR1 amino acid sequence: QNINNF SEQ ID NO: 442VL CDR2 amino acid sequence: AAS SEQ ID NO: 443VL CDR3 amino acid sequence: QQSYGIPW SEQ ID NO: 444 Antibody STIM006VH domain nucleotide sequence: SEQ ID NO: 453CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 454QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFTFSDYF SEQ ID NO: 449VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO: 450VH CDR3 amino acid sequence: ARDHYDGSGIYPLYYYYGLDV SEQ ID NO: 451VL domain nucleotide sequence: SEQ ID NO: 460ATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGCTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO: 459IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRSFGQGTTLEIKVL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO: 456VL CDR2 amino acid sequence: IGS SEQ ID NO: 457VL CDR3 amino acid sequence: MQALQTPRS SEQ ID NO: 458 Antibody STIM007VH domain nucleotide sequence: SEQ ID NO: 467CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 466QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFSLSTTGVG SEQ ID NO: 463VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO: 464VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO: 465VL domain nucleotide sequence: SEQ ID NO: 474GAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC VL domain amino acid sequence: SEQ ID NO: 473EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWPLTFGGGTKVEIKVL CDR1 amino acid sequence: QSVTNY SEQ ID NO: 470VL CDR2 amino acid sequence: DAS SEQ ID NO: 471VL CDR3 amino acid sequence: QHRSNWPLT SEQ ID NO: 472 Antibody STIM008VH domain nucleotide sequence: SEQ ID NO: 481CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 480QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFSLSTSGVG SEQ ID NO: 477VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO: 478VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO: 479VL domain nucleotide sequence: SEQ ID NO: 488GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO: 489EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKVL CDR1 amino acid sequence: QSVTNY SEQ ID NO: 484VL CDR2 amino acid sequence: DAS SEQ ID NO: 485VL CDR3 amino acid sequence: QQRSNWPLT SEQ ID NO: 486 Antibody STIM009VH domain nucleotide sequence: SEQ ID NO: 495CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 494QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFTFSDYY SEQ ID NO: 491VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO: 492VH CDR3 amino acid sequence: ARDFYDILTDSPYFYYGVDV SEQ ID NO: 493VL domain nucleotide sequence: SEQ ID NO: 502GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAVL domain amino acid sequence: SEQ ID NO: 501DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKVL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO: 498VL CDR2 amino acid sequence: IGS SEQ ID NO: 499VL CDR3 amino acid sequence: MQALQTPRT SEQ ID NO: 500

TABLE S1  SEQ ID NOS: 1-342 SEQ  ID NO: Name Description Sequence 1Human PD- NCBI number: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDL1 NP_054862.1 LAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAA(ECD highlighted in LQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVBOLD, cytoplasmic TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTdomain underlined) LRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT HLVILGAILLCLGVALTFIFRLRKGRAMDVKKCGIQDTNSKKQSDTHLEET 2 Cyno PD- NCBI number:MGWSCIILFLVATATGVHSMFTVTVPKDLYVVEYGSNMTIECKFPVEKQ  L1 XP_014973154.1LDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGN (ECD highlighted inAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD BOLD)PVTSEHELTCQAEGYPEAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERT 3 Human PD-Human PD-L1 ECD with MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDL1 His C-terminal His tagLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT HHHHHH 4 Human PD-Human PD-L1 ECD with MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDL1 Fc C-term Fc fusion (inLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAA bold)LQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT IEGREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5 Cyno PD- Cynomolgus PD-L1 ECDMGWSCIILFLVATATGVHSMFTVTVPKDLYVVEYGSNMTIECKFPVEKQ  L1 FLAGwith N-term FLAG tag LDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERT DYKD DDDK 6 Human PD-Human PD-1 full MGWSCIILFLVATATGVHSLDSPDRPWNPPTFSPALLVVTEGDNATFTC 1 Fclength sequence SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGderived from cDNA as RDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPhuman Fc fusion TAHPSPSPRPAGQ KLENLYFQGIEGRMDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSP 7 84G09-Amino acid sequence GFTFDDYA CDRH1 of CDRH1 of 84G09 (IMGT) using IMGT 884G09- Amino acid sequence ISWKSNII CDRH2 of CDRH2 of 84G09 (IMGT)using IMGT 9 84G09- Amino acid sequence ARDITGSGSYGWFDP CDRH3of CDRH3 of 84G09 (IMGT) using IMGT 10 84G09- Amino acid sequence DYAMHCDRH1 of CDRH1 of 84G09 (Kabat) using Kabat 11 84G09-Amino acid sequence GISWKSNIIGYADSVKG CDRH2 of CDRH2 of 84G09 (Kabat)using Kabat 12 84G09- Amino acid sequence DITGSGSYGWFDP CDRH3of CDRH3 of 84G09 (Kabat) using Kabat 13 84G09- Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVS Heavy of V_(H) ofGISWKSNIIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAR chain84G09 (mutations from DITGSGSYGWFDPWGQGTLVTVSS variablegermline are shown region in bold letters) 14 84G09- Nucleic acidCAaGAAAAAGCTTGCCGCCACCATGGAGTTTGGGCTGAGCTGGATTTTC Heavysequence of V_(H) of CTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTACAATTGGTGGAGTchain 84G09 CCGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGC variableAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGACAA regionACTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATAAGTTGGAAGAGTAATATCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTATTGTGCAAGAGATATAACGGGTTCGGGGAGTTATGGCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGAATCT GCTAAAACTCAGCCTCCG 1584G09- Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVS fullof 84G09 heavy chain GISWKSNIIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARheavy (mutations from DITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALchain germline are shownGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS sequencein bold letters) SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK 16 84G09-Nucleic acid GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCAGAT fullsequence of 84G09 CCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCGACGACTACGCheavy heavy chain TATGCACTGGGTGCGACAGACCCCTGGCAAGGGCCTGGAATGGGTGTCCchain GGCATCTCCTGGAAGTCCAACATCATCGGCTACGCCGACTCCGTGAAGG sequenceGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCCTGTACTACTGCGCCAGAGACATCACCGGCTCCGGCTCCTACGGATGGTTCGATCCTTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 17 84G09- Amino acid sequence QSISSYCDRL1 of CDRL1 of 84G09 (IMGT) using IMGT 18 84G09- Amino acid sequenceVAS CDRL2 of CDRL2 of 84G09 (IMGT) using IMGT 19 84G09-Amino acid sequence QQSYSNPIT CDRL3 of CDRL3 of 84G09 (IMGT) using IMGT20 84G09- Amino acid sequence RASQSISSYLN CDRL1 of CDRL1 of 84G09(Kabat) using Kabat 21 84G09- Amino acid sequence VASSLQS CDRL2of CDRL2 of 84G09 (Kabat) using Kabat 22 84G09- Amino acid sequenceQQSYSNPIT CDRL3 of CDRL3 of 84G09 (Kabat) using Kabat 23 84G09-Amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKPLIYLight of VL of 84G09 VASSLQSGVPSSFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPITFchain GQGTRLEIK variable region 24 84G09- Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG Lightsequence of VL of ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTchain 84G09 AAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCCCCTGATCTAT variableGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGTTTCAGTGGCAGTG regionGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTAATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAA 25 84G09- Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKPLIY fullof 84G09 light chain VASSLQSGVPSSFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPITFlight GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 26 84G09- Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG full sequence of 84G09ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTT light light chainAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCCCCTGATCTAT chainGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGTTTCAGTGGCAGTG sequenceGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTAATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 27 1D05-Amino acid sequence GFTFDDYA CDRH1 of CDRH1 of 1D05 (IMGT) using IMGT 281D05- Amino acid sequence ISWIRTGI CDRH2 of CDRH2 of 1D05 (IMGT)using IMGT 29 1D05- Amino acid sequence AKDMKGSGTYGGWFDT CDRH3of CDRH3 of 1D05 (IMGT) using IMGT 30 1D05- Amino acid sequence DYAMHCDRH1 of CDRH1 of 1D05 (Kabat) using Kabat 31 1D05- Amino acid sequenceGISWIRTGIGYADSVKG CDRH2 of CDRH2 of 1D05 (Kabat) using Kabat 32 1D05-Amino acid sequence DMKGSGTYGGWFDT CDRH3 of CDRH3 of 1D05 (Kabat)using Kabat 33 1D05- Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS Heavy of VH of 1D05GISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCAK chain (mutations fromDMKGSGTYGGWFDTWGQGTLVTVSS variable germline are shown regionin bold letters) 34 1D05- Nucleic acidAAGCTTGCCGCCACCATGGAGTTTGGGCTGAGCTGGATTTTCCTTTTGG Heavysequence of V_(H) of CTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGchain 1D05 AGGCTTGGTGCAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCT variableGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGTTCCAG regionGGAAGGGCCTGGAATGGGTCTCAGGCATTAGTTGGATTCGTACTGGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATTTTCAGAGACAACGCCAAGAATTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATGAAGGGTTCGGGGACTTATGGGGGGTGGTTCGACACCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGC 35 1D05- Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS fullof 1D05 heavy chain GISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCAKheavy DMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA chainLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS sequenceSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ  KSLSLSLGK 36 1D05-Nucleic acid GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCAGAT fullsequence of 1D05 CCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCGACGACTACGC heavyheavy chain TATGCACTGGGTGCGACAGGTGCCAGGCAAGGGCCTGGAATGGGTGTCC chainGGCATCTCTTGGATCCGGACCGGCATCGGCTACGCCGACTCTGTGAAGG sequenceGCCGGTTCACCATCTTCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCCTGTACTACTGCGCCAAGGACATGAAGGGCTCCGGCACCTACGGCGGATGGTTCGATACTTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 37 1D05- Amino acid sequence QSISSYCDRL1 of CDRL1 of 1D05 (IMGT) using IMGT 38 1D05- Amino acid sequenceVAS CDRL2 of CDRL2 of 1D05 (IMGT) using IMGT 39 1D05-Amino acid sequence QQSYSTPIT CDRL3 of CDRL3 of 1D05 (IMGT) using IMGT40 1D05- Amino acid sequence RASQSISSYLN CDRL1 of CDRL1 of 1D05 (Kabat)using Kabat 41 1D05- Amino acid sequence VASSLQS CDRL2 of CDRL2 of 1D05(Kabat) using Kabat 42 1D05- Amino acid sequence QQSYSTPIT CDRL3of CDRL3 of 1D05 (Kabat) using Kabat 43 1D05- Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY Light of VL ofVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITF chain1D05 (mutations from GQGTRLEIK variable germline are shown regionin bold letters) 44 1D05- Nucleic acidAAAGCTTGCCGCCACCATGAGGCTCCCTGCTCAGCTTCTGGGGCTCCTG Lightsequence of VL of CTACTCTGGCTCCGAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCchain 1D05 CATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCG variableGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCA regionGGGAAAGCCCCTAAACTCCTGATCTATGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGATCACCTTCGGCCAAGGGACACGTCTGGAGATCAAACGTACGGATGCTGCACCAACT 45 1D05- Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY fullof 1D05 light chain VASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFlight GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 46 1D05- Nucleic acidGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCTTCCGTGGGCG full sequence of 1D05ACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCTCCTACCT light light chainGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTAC chainGTGGCCAGCTCTCTGCAGTCCGGCGTGCCCTCTAGATTCTCCGGCTCTG sequenceGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTCCTACTCCACCCCTATCACCTTCGGCCAGGGCACCCGGCTGGAAATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 47 MutatedAmino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ A PGKGLEWVS1D05-HC of 1D05 heavy chainGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCAK mutant 1with V to A back- DMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAmutation in LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSframework region to SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE LAGA PSVFLgermline highlighted FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPwith IgG1 disabled REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK(LAGA) constant GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN regionNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ  KSLSLSLGK 48 MutatedAmino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS1D05-HC of 1D05 heavy chain GISWIRTGIGYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAK mutant 2 with F to S back-DMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA mutation inLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS framework region toSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE LAGA PSVFL germline highlightedFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP with IgG1 disabledREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (LAGA) constantGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN regionNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ  KSLSLSLGK 49 MutatedAmino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS1D05-HC of 1D05 heavy chainGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCAK mutant 3with ELLG to -PVA DMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAback-mutation in LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSconstant region to SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP -  germline highlighted PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK 50Mutated Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY 1D05-LCof 1D05 kappa light A ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFmutant 1 chain with V to AGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  back-mutation inWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV CDRL2 to germlineTHQGLSSPVTKSFNRGEC highlighted 51 Mutated Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL F IY 1D05-LCof 1D05 kappa light VASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFmutant 2 chain with L to FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  back-mutation inWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV framework toTHQGLSSPVTKSFNRGEC germline highlighted 52 411B08- Amino acid sequenceGFTFSSYW CDRH1 of CDRH1 of 411B08 (IMGT) using IMGT 53 411B08-Amino acid sequence IKEDGSEK CDRH2 of CDRH2 of 411B08 (IMGT) using IMGT54 411B08- Amino acid sequence ARNRLYSDFLDN CDRH3 of CDRH3 of 411B08(IMGT) using IMGT 55 411B08- Amino acid sequence SYWMS CDRH1of CDRH1 of 411B08 (Kabat) using Kabat 56 411B08- Amino acid sequenceNIKEDGSEKYYVDSVKG CDRH2 of CDRH2 of 411B08 (Kabat) using Kabat 57411B08- Amino acid sequence NRLYSDFLDN CDRH3 of CDRH3 of 411B08 (Kabat)using Kabat 58 411B08- Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA Heavyof V_(H) of 411B08 NIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCARchain NRLYSDFLDNWGQGTLVTVSS variable region 59 411B08- Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT Heavysequence of V_(H) of CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGchain 411B08 GATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCC variableAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGAAGG regionGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCAG 60411B08- Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA full of 411B08 heavyNIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCAR heavy chainNRLYSDFLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL chainVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG sequenceTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 61 411B08-Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT fullsequence of 411B08 CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGheavy heavy chain GATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCchain AACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGAAGG sequenceGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 62 411B08- Amino acid sequence QGVSSW CDRL1of CDRL1 of 411B08 (IMGT) using IMGT 63 411B08- Amino acid sequence GASCDRL2 of CDRL2 of 411B08 (IMGT) using IMGT 64 411B08-Amino acid sequence QQANSIPFT CDRL3 of CDRL3 of 411B08 (IMGT) using IMGT65 411B08- Amino acid sequence RASQGVSSWLA CDRL1 of CDRL1 of 411B08(Kabat) using Kabat 66 411B08- Amino acid sequence GASSLQS CDRL2of CDRL2 of 411B08 (Kabat) using Kabat 67 411B08- Amino acid sequenceQQANSIPFT CDRL3 of CDRL3 of 411B08 (Kabat) using Kabat 68 411B08-Amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIYLight of VL of 411B08 GASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFTFchain GPGTKVDIK variable region 69 411B08- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAG Lightsequence of V_(L) of ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGTTchain 411B08 AGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTAT variableGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTG regionGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 70 411B08- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIY full of 411B08 lightGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFTF light chainGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 71 411B08- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAG fullsequence of 411B08 ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGTTlight light chain AGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTATchain GGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTG sequenceGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 72 411C04-Amino acid sequence GFTFSSYW CDRH1 of CDRH1 of 411C04 (IMGT) using IMGT73 411C04- Amino acid sequence IKEDGSEK CDRH2 of CDRH2 of 411C04 (IMGT)using IMGT 74 411C04- Amino acid sequence ARVRLYSDFLDY CDRH3of CDRH3 of 411C04 (IMGT) using IMGT 75 411C04- Amino acid sequenceSYWMS CDRH1 of CDRH1 of 411C04 (Kabat) using Kabat 76 411C04-Amino acid sequence NIKEDGSEKYYVDSLKG CDRH2 of CDRH2 of 411C04 (Kabat)using Kabat 77 411C04- Amino acid sequence VRLYSDFLDY CDRH3of CDRH3 of 411C04 (Kabat) using Kabat 78 411C04- Amino acid sequenceEVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA Heavyof V_(H) of 411C04 NIKEDGSEKYYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCARchain VRLYSDFLDYWGQGTLVTVSS variable region 79 411C04- Nucleic acidGAGGTGCAGCTGGTGGACTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT Heavysequence of V_(H) of CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGchain 411C04 GATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTGGCC variableAACATAAAAGAAGATGGAAGTGAGAAATACTATGTAGACTCTTTGAAGG regionGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAGTTCGACTCTACAGTGACTTCCTTGACTACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCAG 80411C04- Amino acid sequenceEVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA full of 411C04 heavyNIKEDGSEKYYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCAR heavy chainVRLYSDFLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL chainVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG sequenceTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 81 411C04-Nucleic acid GAGGTGCAGCTGGTGGACTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT fullsequence of 411C04 CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGheavy heavy chain GATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTGGCCchain AACATAAAAGAAGATGGAAGTGAGAAATACTATGTAGACTCTTTGAAGG sequenceGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAGTTCGACTCTACAGTGACTTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 82 411C04- Amino acid sequence QGVSSW CDRL1of CDRL1 of 411C04 (IMGT) using IMGT 83 411C04- Amino acid sequence GASCDRL2 of CDRL2 of 411C04 (IMGT) using IMGT 84 411C04-Amino acid sequence QQANSIPFT CDRL3 of CDRL3 of 411C04 (IMGT) using IMGT85 411C04- Amino acid sequence RASQGVSSWLA CDRL1 of CDRL1 of 411C04(Kabat) using Kabat 86 411C04- Amino acid sequence GASSLQS CDRL2of CDRL2 of 411C04 (Kabat) using Kabat 87 411C04- Amino acid sequenceQQANSIPFT CDRL3 of CDRL3 of 411C04 (Kabat) using Kabat 88 411C04-Amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIYLight of V_(L) of 411C04GASSLQSGVPSRFSGSGSGTEFILSISSLQPEDFATYYCQQANSIPFTF chain GPGTKVDIKvariable region 89 411C04- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAG Lightsequence of V_(L) of ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGTTGGTTchain 411C04 AGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTAT variableGGTGCCTCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTG regionGATCTGGGACAGAGTTCATTCTCAGCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 90 411C04- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIY full of 411C04 lightGASSLQSGVPSRFSGSGSGTEFILSISSLQPEDFATYYCQQANSIPFTF light chainGPGTKVDIKRTVVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE sequenceTHQGLSSPVTKSFNRGEC 91 411C04- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAG fullsequence of 411C04 ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGTTGGTTlight light chain AGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTATchain GGTGCCTCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTG sequenceGATCTGGGACAGAGTTCATTCTCAGCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 92 411D07-Amino acid sequence GGSIISSDW CDRH1 of CDRH1 of 411D07 (IMGT) using IMGT93 411D07- Amino acid sequence IFHSGRT CDRH2 of CDRH2 of 411D07 (IMGT)using IMGT 94 411D07- Amino acid sequence ARDGSGSY CDRH3of CDRH3 of 411D07 (IMGT) using IMGT 95 411D07- Amino acid sequenceSSDWWN CDRH1 of CDRH1 of 411D07 (Kabat) using Kabat 96 411D07-Amino acid sequence EIFHSGRTNYNPSLKS CDRH2 of CDRH2 of 411D07 (Kabat)using Kabat 97 411D07- Amino acid sequence DGSGSY CDRH3of CDRH3 of 411D07 (Kabat) using Kabat 98 411D07- Amino acid sequenceQVQLQESGPGLVKPSGTLSLTCIVSGGSIISSDWWNWVRQPPGKGLEWI Heavyof V_(H) of 411D07 GEIFHSGRTNYNPSLKSRVTISIDKSKNQFSLRLSSVTAADTAVYYCARchain DGSGSYWGQGTLVTVSS variable region 99 411D07- Nucleic acidCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGA Heavysequence of V_(H) of CCCTGTCCCTCACCTGCATTGTCTCTGGTGGCTCCATCATCAGTAGTGAchain 411D07 CTGGTGGAATTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT variableGGAGAAATCTTTCATAGTGGGAGGACCAACTACAACCCGTCCCTCAAGA regionGTCGAGTCACCATATCAATAGACAAGTCCAAGAATCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCT CAG 100 411D07-Amino acid sequence QVQLQESGPGLVKPSGTLSLTCIVSGGSIISSDWWNWVRQPPGKGLEWIfull of 411D07 heavy GEIFHSGRTNYNPSLKSRVTISIDKSKNQFSLRLSSVTAADTAVYYCARheavy chain DGSGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY chainFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY sequenceICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 101 411D07-Nucleic acid CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGA fullsequence of 411D07 CCCTGTCCCTCACCTGCATTGTCTCTGGTGGCTCCATCATCAGTAGTGAheavy heavy chain CTGGTGGAATTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTchain GGAGAAATCTTTCATAGTGGGAGGACCAACTACAACCCGTCCCTCAAGA sequenceGTCGAGTCACCATATCAATAGACAAGTCCAAGAATCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG AGCCCCGGCAAG 102411D07- Amino acid sequence QSVLYSSNNKNY CDRL1 of CDRL1 of 411D07 (IMGT)using IMGT 103 411D07- Amino acid sequence WAS CDRL2 of CDRL2 of 411D07(IMGT) using IMGT 104 411D07- Amino acid sequence QQYYSNRS CDRL3of CDRL3 of 411D07 (IMGT) using IMGT 105 411D07- Amino acid sequenceKSSQSVLYSSNNKNYLA CDRL1 of CDRL1 of 411D07 (Kabat) using Kabat 106411D07- Amino acid sequence WASTRES CDRL2 of CDRL2 of 411D07 (Kabat)using Kabat 107 411D07- Amino acid sequence QQYYSNRS CDRL3of CDRL3 of 411D07 (Kabat) using Kabat 108 411D07- Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKSGQP Lightof V_(L) of 411D07 PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQTEDVAVYYCQQYYchain SNRSFGQGTKLEIK variable region 109 411D07- Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCG Lightsequence of V_(L) of AGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCchain 411D07 CAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAATCAGGACAGCCT variableCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTG regionACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTAATCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC 110 411D07-Amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKSGQPfull of 411D07 light PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQTEDVAVYYCQQYYlight chain SNRSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR chainEAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV sequenceYACEVTHQGLSSPVTKSFNRGEC 111 411D07- Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCG fullsequence of 411D07 AGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTClight light chain CAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAATCAGGACAGCCTchain CCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTG sequenceACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTAATCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGT CTTTCAACCGGGGCGAGTGT112 385F01- Amino acid sequence GFTFSSYW CDRH1 of CDRH1 of 385F01 (IMGT)using IMGT 113 385F01- Amino acid sequence IKEDGSEK CDRH2of CDRH2 of 385F01 (IMGT) using IMGT 114 385F01- Amino acid sequenceARNRLYSDFLDN CDRH3 of CDRH3 of 385F01 (IMGT) using IMGT 115 385F01-Amino acid sequence SYWMS CDRH1 of CDRH1 of 385F01 (Kabat) using Kabat116 385F01- Amino acid sequence NIKEDGSEKYYVDSVKG CDRH2of CDRH2 of 385F01 (Kabat) using Kabat 117 385F01- Amino acid sequenceNRLYSDFLDN CDRH3 of CDRH3 of 385F01 (Kabat) using Kabat 118 385F01-Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVAHeavy of V_(H) of 385F01NIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCAR chainNRLYSDFLDNWGQGTLVTVSS variable region 119 385F01- Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT Heavysequence of V_(H) of CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGchain 385F01 GATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCC variableAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGAAGG regionGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCAG 120385F01- Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA full of 385F01 heavyNIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCAR heavy chainNRLYSDFLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL chainVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG sequenceTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 121 385F01-Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT fullsequence of 385F01 CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGheavy heavy chain GATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCchain AACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGAAGG sequenceGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 122 385F01- Amino acid sequence QGVSSW CDRL1of CDRL1 of 385F01 (IMGT) using IMGT 123 385F01- Amino acid sequence GASCDRL2 of CDRL2 of 385F01 (IMGT) using IMGT 124 385F01-Amino acid sequence QQANSIPFT CDRL3 of CDRL3 of 385F01 (IMGT) using IMGT125 385F01- Amino acid sequence RASQGVSSWLA CDRL1 of CDRL1 of 385F01(Kabat) using Kabat 126 385F01- Amino acid sequence GASSLQS CDRL2of CDRL2 of 385F01 (Kabat) using Kabat 127 385F01- Amino acid sequenceQQANSIPFT CDRL3 of CDRL3 of 385F01 (Kabat) using Kabat 128 385F01-Amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIYLight of V_(L) of 385F01GASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFTF chain GPGTKVDIKvariable region 129 385F01- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAG Lightsequence of V_(L) of ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGTTchain 385F01 AGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTAT variableGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTG regionGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 130 385F01- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIY full of 385F01 lightGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFTF light chainGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 131 385F01- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAG fullsequence of 385F01 ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGTTlight light chain AGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTATchain GGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTG sequenceGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 132 413D08-Amino acid sequence GFTFRIYG CDRH1 of CDRH1 of 413D08 (IMGT) using IMGT133 413D08- Amino acid sequence IWYDGSNK CDRH2 of CDRH2 of 413D08 (IMGT)using IMGT 134 413D08- Amino acid sequence ARDMDYFGMDV CDRH3of CDRH3 of 413D08 (IMGT) using IMGT 135 413D08- Amino acid sequenceIYGMH CDRH1 of CDRH1 of 413D08 (Kabat) using Kabat 136 413D08-Amino acid sequence VIWYDGSNKYYADSVKG CDRH2 of CDRH2 of 413D08 (Kabat)using Kabat 137 413D08- Amino acid sequence DMDYFGMDV CDRH3of CDRH3 of 413D08 (Kabat) using Kabat 138 413D08- Amino acid sequenceQVQLVESGGGVVQPGRSLRLSCAASGFTFRIYGMHWVRQAPGKGLEWVA Heavyof V_(H) of 413D08 VIWYDGSNKYYADSVKGRFTISRDNSDNTLYLQMNSLRAEDTAVYYCARchain DMDYFGMDVWGQGTTVTVSS variable region 139 413D08- Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGT Heavysequence of V_(H) of CCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCCGTATTTATGGchain 413D08 CATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCA variableGTTATATGGTATGATGGAAGTAATAAATACTATGCTGACTCCGTGAAGG regionGCCGATTCACCATCTCCAGAGACAATTCCGACAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATATGGACTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCCTCAG 140413D08- Amino acid sequenceQVQLVESGGGVVQPGRSLRLSCAASGFTFRIYGMHWVRQAPGKGLEWVA full of 413D08 heavyVIWYDGSNKYYADSVKGRFTISRDNSDNTLYLQMNSLRAEDTAVYYCAR heavy chainDMDYFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV chainKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT sequenceQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 141 413D08-Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGT fullsequence of 413D08 CCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCCGTATTTATGGheavy heavy chain CATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAchain GTTATATGGTATGATGGAAGTAATAAATACTATGCTGACTCCGTGAAGG sequenceGCCGATTCACCATCTCCAGAGACAATTCCGACAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATATGGACTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCC CTGTCCCTGAGCCCCGGCAAG142 413D08- Amino acid sequence QGIRND CDRL1 of CDRL1 of 413D08 (IMGT)using IMGT 143 413D08- Amino acid sequence AAS CDRL2 of CDRL2 of 413D08(IMGT) using IMGT 144 413D08- Amino acid sequence LQHNSYPRT CDRL3of CDRL3 of 413D08 (IMGT) using IMGT 145 413D08- Amino acid sequenceRASQGIRNDLG CDRL1 of CDRL1 of 413D08 (Kabat) using Kabat 146 413D08-Amino acid sequence AASSLQS CDRL2 of CDRL2 of 413D08 (Kabat) using Kabat147 413D08- Amino acid sequence LQHNSYPRT CDRL3 of CDRL3 of 413D08(Kabat) using Kabat 148 413D08- Amino acid sequenceDLQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIY Lightof V_(L) of 413D08 AASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFchain GQGTKVEIK variable region 149 413D08- Nucleic acidGACCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG Lightsequence of V_(L) of ACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTchain 413D08 AGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTAT variableGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG regionGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC 150 413D08- Amino acid sequenceDLQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIY full of 413D08 lightAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTF light chainGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 151 413D08- Nucleic acidGACCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG fullsequence of 413D08 ACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTlight light chain AGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATchain GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG sequenceGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 152 386H03-Amino acid sequence GGSISSSDW CDRH1 of CDRH1 of 386H03 (IMGT) using IMGT153 386H03- Amino acid sequence IFHSGNT CDRH2 of CDRH2 of 386H03 (IMGT)using IMGT 154 386H03- Amino acid sequence VRDGSGSY CDRH3of CDRH3 of 386H03 (IMGT) using IMGT 155 386H03- Amino acid sequenceSSDWWS CDRH1 of CDRH1 of 386H03 (Kabat) using Kabat 156 386H03-Amino acid sequence EIFHSGNTNYNPSLKS CDRH2 of CDRH2 of 386H03 (Kabat)using Kabat 157 386H03- Amino acid sequence DGSGSY CDRH3of CDRH3 of 386H03 (Kabat) using Kabat 158 386H03- Amino acid sequenceQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWI Heavyof V_(H) of 386H03 GEIFHSGNTNYNPSLKSRVTISVDKSKNQISLRLNSVTAADTAVYYCVRchain DGSGSYWGQGTLVTVSS variable region 159 386H03- Nucleic acidCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGA Heavysequence of V_(H) of CCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTGAchain 386H03 CTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT variableGGGGAAATCTTTCATAGTGGGAACACCAACTACAACCCGTCCCTCAAGA regionGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGATCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGTGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCT CAG 160 386H03-Amino acid sequence QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWIfull of 386H03 heavy GEIFHSGNTNYNPSLKSRVTISVDKSKNQISLRLNSVTAADTAVYYCVRheavy chain DGSGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY chainFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY sequenceICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 161 386H03-Nucleic acid CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGA fullsequence of 386H03 CCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTGAheavy heavy chain CTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTchain GGGGAAATCTTTCATAGTGGGAACACCAACTACAACCCGTCCCTCAAGA sequenceGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGATCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGTGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG AGCCCCGGCAAG 162386H03- Amino acid sequence QSVLYSSNNKNY CDRL1 of CDRL1 of 386H03 (IMGT)using IMGT 163 386H03- Amino acid sequence WAS CDRL2 of CDRL2 of 386H03(IMGT) using IMGT 164 386H03- Amino acid sequence QQYYSTRS CDRL3of CDRL3 of 386H03 (IMGT) using IMGT 165 386H03- Amino acid sequenceKSSQSVLYSSNNKNYLA CDRL1 of CDRL1 of 386H03 (Kabat) using Kabat 166386H03- Amino acid sequence WASTRES CDRL2 of CDRL2 of 386H03 (Kabat)using Kabat 167 386H03- Amino acid sequence QQYYSTRS CDRL3of CDRL3 of 386H03 (Kabat) using Kabat 168 386H03- Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP Lightof V_(L) of 386H03 PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYchain STRSFGQGTKLEIK variable region 169 386H03- Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCG Lightsequence of V_(L) of AGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCchain 386H03 CAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCT variableCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTG regionACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC 170 386H03-Amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPfull of 386H03 light PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYlight chain STRSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR chainEAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV sequenceYACEVTHQGLSSPVTKSFNRGEC 171 386H03- Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCG fullsequence of 386H03 AGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTClight light chain CAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTchain CCTAAACTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTG sequenceACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGT CTTTCAACCGGGGCGAGTGT172 389A03- Amino acid sequence GGSISSSSYY CDRH1 of CDRH1 of 389A03(IMGT) using IMGT 173 389A03- Amino acid sequence IYSTGYT CDRH2of CDRH2 of 389A03 (IMGT) using IMGT 174 389A03- Amino acid sequenceAISTAAGPEYFHR CDRH3 of CDRH3 of 389A03 (IMGT) using IMGT 175 389A03-Amino acid sequence SSSYYCG CDRH1 of CDRH1 of 389A03 (Kabat) using Kabat176 389A03- Amino acid sequence SIYSTGYTYYNPSLKS CDRH2of CDRH2 of 389A03 (Kabat) using Kabat 177 389A03- Amino acid sequenceSTAAGPEYFHR CDRH3 of CDRH3 of 389A03 (Kabat) using Kabat 178 389A03-Amino acid sequence QLQESGPGLVKPSETLSLTCTVSGGSISSSSYYCGWIRQPPGKGLDWIGHeavy of V_(H) of 389A03SIYSTGYTYYNPSLKSRVTISIDTSKNQFSCLILTSVTAADTAVYYCAI chainSTAAGPEYFHRWGQGTLVTVSS variable region 179 389A03- Nucleic acidCAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCGGAGACCCTGT Heavysequence of V_(H) of CCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTATTAchain 389A03 CTGCGGCTGGATCCGCCAGCCCCCTGGGAAGGGGCTGGACTGGATTGGG variableAGTATCTATTCTACTGGGTACACCTACTACAACCCGTCCCTCAAGAGTC regionGAGTCACCATTTCCATAGACACGTCCAAGAACCAGTTCTCATGCCTGATACTGACCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGATAAGTACAGCAGCTGGCCCTGAATACTTCCATCGCTGGGGCCAGGGCACCC TGGTCACCGTCTCCTCAG 180389A03- Amino acid sequenceQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYCGWIRQPPGKGLDWIG full of 389A03 heavySIYSTGYTYYNPSLKSRVTISIDTSKNQFSCLILTSVTAADTAVYYCAI heavy chainSTAAGPEYFHRWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC chainLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL sequenceGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ  KSLSLSPGK 181 389A03-Nucleic acid CAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCGGAGACCCTGT fullsequence of 389A03 CCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTATTAheavy heavy chain CTGCGGCTGGATCCGCCAGCCCCCTGGGAAGGGGCTGGACTGGATTGGGchain AGTATCTATTCTACTGGGTACACCTACTACAACCCGTCCCTCAAGAGTC sequenceGAGTCACCATTTCCATAGACACGTCCAAGAACCAGTTCTCATGCCTGATACTGACCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGATAAGTACAGCAGCTGGCCCTGAATACTTCCATCGCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 182 389A03- Amino acid sequence QSVLYSSNSKNFCDRL1 of CDRL1 of 389A03 (IMGT) using IMGT 183 389A03-Amino acid sequence WAS CDRL2 of CDRL2 of 389A03 (IMGT) using IMGT 184389A03- Amino acid sequence QQYYSTPRT CDRL3 of CDRL3 of 389A03 (IMGT)using IMGT 185 389A03- Amino acid sequence KSSQSVLYSSNSKNFLA CDRL1of CDRL1 of 389A03 (Kabat) using Kabat 186 389A03- Amino acid sequenceWASTRGS CDRL2 of CDRL2 of 389A03 (Kabat) using Kabat 187 389A03-Amino acid sequence QQYYSTPRT CDRL3 of CDRL3 of 389A03 (Kabat)using Kabat 188 389A03- Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNFLAWYQQKPGQP Lightof V_(L) of 389A03 PKLFIYWASTRGSGVPDRISGSGSGTDFNLTISSLQAEDVAVYYCQQYYchain STPRTFGQGTKVEIK variable region 189 389A03- Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCG Lightsequence of V_(L) of AGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCchain 389A03 CAACAGTAAGAACTTCTTAGCTTGGTACCAGCAGAAACCGGGACAGCCT variableCCTAAGCTGTTCATTTACTGGGCATCTACCCGGGGATCCGGGGTCCCTG regionACCGAATCAGTGGCAGCGGGTCTGGGACAGATTTCAATCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAAC 190 389A03-Amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNFLAWYQQKPGQPfull of 389A03 light PKLFIYWASTRGSGVPDRISGSGSGTDFNLTISSLQAEDVAVYYCQQYYlight chain STPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP chainREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK sequenceVYACEVTHQGLSSPVTKSFNRGEC 191 389A03- Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCG fullsequence of 389A03 AGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTClight light chain CAACAGTAAGAACTTCTTAGCTTGGTACCAGCAGAAACCGGGACAGCCTchain CCTAAGCTGTTCATTTACTGGGCATCTACCCGGGGATCCGGGGTCCCTG sequenceACCGAATCAGTGGCAGCGGGTCTGGGACAGATTTCAATCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 192 Human IGHG*01 Heavy Chaingcttccaccaagggcccatccgtcttccccctggcgccctgctccagga IgG4 & Constantgcacctccgagagcacagccgccctgggctgcctggtcaaggactactt heavy IGHG4*04 Regionccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggc chain Nucleotidegtgcacaccttcccggctgtcctacagtcctcaggactctactccctca constant Sequencegcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacac region #1ctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaa a 193 Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG ConstantVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV RegionESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS Amino AcidQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG SequenceKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 194 Human IGHG*02  Heavy Chaingcttccaccaagggcccatccgtcttccccctggcgccctgctccagga IgG4 Constantgcacctccgagagcacagccgccctgggctgcctggtcaaggactactt heavy Regionccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggc chain Nucleotidegtgcacaccttcccggctgtcctacagtcctcaggactctactccctca constant Sequencegcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacac region #2ctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccgtgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctctgggtaa a 195 Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG ConstantVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV RegionESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS Amino AcidQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNG SequenceKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 196 Human IGHG*0 3 Heavy Chaingcttccaccaagggcccatccgtcttccccctggcgccctgctccagga IgG4 Constantgcacctccgagagcacagccgccctgggctgcctggtcaaggactactt heavy Regionccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggc chain Nucleotidegtgcacaccttcccggctgtcctacagtcctcaggactctactccctca constant Sequencegcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacac region #3ctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcaggaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctctgggtaa a 197 Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG ConstantVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV RegionESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS Amino AcidQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG SequenceKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 198 IgG4 -IgG4- Heavy Chaingcctccaccaagggcccatccgtcttccccctggcgccctgctccagga heavy PE Constantgcacctccgagagcacggccgccctgggctgcctggtcaaggactactt chain Regionccccgaaccagtgacggtgtcgtggaactcaggcgccctgaccagcggc constant Nucleotidegtgcacaccttcccggctgtcctacagtcctcaggactctactccctca region- Sequence-gcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacac IgG4-PE Syntheticctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagtt Version Agagtccaaatatggtcccccatgcccaccatgcccagcgcctgaatttgaggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggatccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaa a 199 IgG4 Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG heavy ConstantVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV chain RegionESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS constant Amino AcidQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG region- Sequence-KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL IgG4-PE Encoded byTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SyntheticSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Version A, B & C(Two residuesthat differ from the wild-type sequence are identified in bold) 200 IgG4Heavy Chain Gcctccaccaagggacctagcgtgttccctctcgccccctgttccaggt heavyConstant ccacaagcgagtccaccgctgccctcggctgtctggtgaaagactactt chain Regiontcccgagcccgtgaccgtctcctggaatagcggagccctgacctccggc constant Nucleotidegtgcacacatttcccgccgtgctgcagagcagcggactgtatagcctga region- Sequence-gcagcgtggtgaccgtgcccagctccagcctcggcaccaaaacctacac IgG4-PE Syntheticctgcaacgtggaccacaagccctccaacaccaaggtggacaagcgggtg Version Bgagagcaagtacggccccccttgccctccttgtcctgcccctgagttcgagggaggaccctccgtgttcctgtttccccccaaacccaaggacaccctgatgatctcccggacacccgaggtgacctgtgtggtcgtggacgtcagccaggaggaccccgaggtgcagttcaactggtatgtggacggcgtggaggtgcacaatgccaaaaccaagcccagggaggagcagttcaattccacctacagggtggtgagcgtgctgaccgtcctgcatcaggattggctgaacggcaaggagtacaagtgcaaggtgtccaacaagggactgcccagctccatcgagaagaccatcagcaaggctaagggccagccgagggagccccaggtgtataccctgcctcctagccaggaagagatgaccaagaaccaagtgtccctgacctgcctggtgaagggattctacccctccgacatcgccgtggagtgggagagcaatggccagcccgagaacaactacaaaacaacccctcccgtgctcgatagcgacggcagcttctttctctacagccggctgacagtggacaagagcaggtggcaggagggcaacgtgttctcctgttccgtgatgcacgaggccctgcacaatcactacacccagaagagcctctccctgtccctgggcaa g 201 IgG4 Heavy Chaingccagcaccaagggcccttccgtgttccccctggccccttgcagcagga heavy Constantgcacctccgaatccacagctgccctgggctgtctggtgaaggactactt chain Regiontcccgagcccgtgaccgtgagctggaacagcggcgctctgacatccggc constant Nucleotidegtccacacctttcctgccgtcctgcagtcctccggcctctactccctgt region- Sequence-cctccgtggtgaccgtgcctagctcctccctcggcaccaagacctacac IgG4-PE Syntheticctgtaacgtggaccacaaaccctccaacaccaaggtggacaaacgggtc Version Cgagagcaagtacggccctccctgccctccttgtcctgcccccgagttcgaaggcggacccagcgtgttcctgttccctcctaagcccaaggacaccctcatgatcagccggacacccgaggtgacctgcgtggtggtggatgtgagccaggaggaccctgaggtccagttcaactggtatgtggatggcgtggaggtgcacaacgccaagacaaagccccgggaagagcagttcaactccacctacagggtggtcagcgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtacaagtgcaaggtcagcaataagggactgcccagcagcatcgagaagaccatctccaaggctaaaggccagccccgggaacctcaggtgtacaccctgcctcccagccaggaggagatgaccaagaaccaggtgagcctgacctgcctggtgaagggattctacccttccgacatcgccgtggagtgggagtccaacggccagcccgagaacaattataagaccacccctcccgtcctcgacagcgacggatccttctttctgtactccaggctgaccgtggataagtccaggtggcaggaaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaatcactacacccagaagtccctgagcctgtccctgggaaa g 202 IgG4 Heavy Chaingcctccaccaagggcccatccgtcttccccctggcgccctgctccagga heavy Constantgcacctccgagagcacggccgccctgggctgcctggtcaaggactactt chain Regionccccgaaccagtgacggtgtcgtggaactcaggcgccctgaccagcggc constant Nucleotidegtgcacaccttcccggctgtcctacagtcctcaggactctactccctca region Sequence-gcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacac Syntheticctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagtt Version Dgagtccaaatatggtcccccatgcccaccatgcccagcgcctccagttgcggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggatccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaa a 203 Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG ConstantVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV RegionESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS Amino AcidQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG Sequence-KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL encoded byTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SyntheticSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Version D 204 Disabled Disabled Heavy Chain gcctccaccaagggcccatcggtcttccccctggcaccctcctccaaga HumanIGHG1 Constant gcacctctgggggcacagcggccctgggctgcctggtcaaggactactt IgG1Region ccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggc heavyNucleotide gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca chainSequence gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat constantctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtg regiongagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcgcgggggcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc tccgggtaaa 205Heavy Chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG ConstantVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV RegionEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVV Amino AcidDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW SequenceLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ  (TwoVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT residuesVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK that differ from the wild-typesequence are identified in bold) 206 Human Cκ IGKC*01 Cκ Lightcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagc constant Chainagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttcta region Constantcccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtcc Regionggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacct Nucleotideactccctgtcctccaccctgaccctgtccaaggccgactacgagaagca Sequencecaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 207 Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS ChainGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Constant TKSFNRGECRegion Amino Acid Sequence 208 Human Cκ IGKC*02 Cκ Lightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc constant Chainagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcta region Constanttcccagagaggccaaagtacagtggaaggtggataacgccctccaatcg Regionggtaactcccaggagagtgtcacagagcaggagagcaaggacagcacct Nucleotideacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaaca Sequencecaaagtctacgccggcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 209 Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS ChainGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSPV Constant TKSFNRGECRegion Amino Acid Sequence 210 Human Cκ IGKC*03 Cκ Lightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc constant Chainagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcta region Constanttcccagagaggccaaagtacagcggaaggtggataacgccctccaatcg Regionggtaactcccaggagagtgtcacagagcaggagagcaaggacagcacct Nucleotideacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaaca Sequencecaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 211 Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQRKVDNALQS ChainGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Constant TKSFNRGECRegion Amino Acid Sequence 212 Human Cκ IGKC*04 Cκ Lightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc constant Chainagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcta region Constanttcccagagaggccaaagtacagtggaaggtggataacgccctccaatcg Regionggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacct Nucleotideacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaaca Sequencecaaactctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 213 Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS ChainGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPV Constant TKSFNRGECRegion Amino Acid Sequence 214 Human Cκ IGKC*05 Cκ Lightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc constant Chainagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcta region Constanttcccagagaggccaaagtacagtggaaggtggataacgccctccaatcg Regionggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacct Nucleotideacagcctcagcaacaccctgacgctgagcaaagcagactacgagaaaca Sequencecaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc 215 Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS ChainGNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPV Constant TKSFNRGECRegion Amino Acid Sequence 216 Human Cλ IGCA1*01 Cλ Lightcccaaggccaaccccacggtcactctgttcccgccctcctctgaggagc constant Chaintccaagccaacaaggccacactagtgtgtctgatcagtgacttctaccc region Constantgggagctgtgacagtggcttggaaggcagatggcagccccgtcaaggcg Regionggagtggagacgaccaaaccctccaaacagagcaacaacaagtacgcgg Nucleotideccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaag Sequencectacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtg gcccctacagaatgttca 217Cλ Light PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKA ChainGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV Constant APTECS RegionAmino Acid Sequence 218 Human Cλ IGCA1*02 Cλ Lightggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctg constant Chainaggagctccaagccaacaaggccacactagtgtgtctgatcagtgactt region Constantctacccgggagctgtgacagtggcctggaaggcagatggcagccccgtc Regionaaggcgggagtggagaccaccaaaccctccaaacagagcaacaacaagt Nucleotideacgcggccagcagctacctgagcctgacgcccgagcagtggaagtccca Sequencecagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 219 Cλ LightGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPV ChainKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK Constant TVAPTECSRegion Amino Acid Sequence 220 Human Cλ IGCA2*01 Cλ Lightggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctg constant Chainaggagctccaagccaacaaggccacactagtgtgtctgatcagtgactt region Constantctacccgggagctgtgacagtggcctggaaggcagatggcagccccgtc Regionaaggcgggagtggagaccaccaaaccctccaaacagagcaacaacaagt Nucleotideacgcggccagcagctacctgagcctgacgcccgagcagtggaagtccca Sequence-cagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaag Version Aacagtggcccctacagaatgttca 221 Cλ Lightggccagcctaaggccgctccttctgtgaccctgttccccccatcctccg Chainaggaactgcaggctaacaaggccaccctcgtgtgcctgatcagcgactt Constantctaccctggcgccgtgaccgtggcctggaaggctgatagctctcctgtg Regionaaggccggcgtggaaaccaccaccccttccaagcagtccaacaacaaat Nucleotideacgccgcctcctcctacctgtccctgacccctgagcagtggaagtccca Sequence-ccggtcctacagctgccaagtgacccacgagggctccaccgtggaaaag Version Baccgtggctcctaccgagtgctcc 222 Cλ Lightggccagcctaaagctgcccccagcgtcaccctgtttcctccctccagcg Chainaggagctccaggccaacaaggccaccctcgtgtgcctgatctccgactt Constantctatcccggcgctgtgaccgtggcttggaaagccgactccagccctgtc Regionaaagccggcgtggagaccaccacaccctccaagcagtccaacaacaagt Nucleotideacgccgcctccagctatctctccctgacccctgagcagtggaagtccca Sequence-ccggtcctactcctgtcaggtgacccacgagggctccaccgtggaaaag Version Caccgtcgcccccaccgagtgctcc 223 Cλ LightGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPV ChainKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK Constant TVAPTECSRegion Amino Acid Sequence- Encoded by Version A, B & C 224 Human CλIGCA2*02 Cλ Light ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgconstant & Chain aggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttregion IGLC2*03 Constantctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtc Regionaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagt Nucleotideacgcggccagcagctatctgagcctgacgcctgagcagtggaagtccca Sequencecagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 225 Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV ChainKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK Constant TVAPTECSRegion Amino Acid Sequence 226 Human Cλ IGCA3*01 Cλ Lightcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagc constant Chainttcaagccaacaaggccacactggtgtgtctcataagtgacttctaccc region Constantgggagccgtgacagttgcctggaaggcagatagcagccccgtcaaggcg Regionggggtggagaccaccacaccctccaaacaaagcaacaacaagtacgcgg Nucleotideccagcagctacctgagcctgacgcctgagcagtggaagtcccacaaaag Sequencectacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtt gcccctacggaatgttca 227Cλ Light PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA ChainGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTV Constant APTECS RegionAmino Acid Sequence 228 Human Cλ IGCA3*02 Cλ Lightggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctg constant Chainaggagcttcaagccaacaaggccacactggtgtgtctcataagtgactt region Constantctacccggggccagtgacagttgcctggaaggcagatagcagccccgtc Regionaaggcgggggtggagaccaccacaccctccaaacaaagcaacaacaagt Nucleotideacgcggccagcagctacctgagcctgacgcctgagcagtggaagtccca Sequencecaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacggaatgttca 229 Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGPVTVAWKADSSPV ChainKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEK Constant TVAPTECSRegion Amino Acid Sequence 230 Human Cλ IGCA3*03 Cλ Lightggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctg constant Chainaggagcttcaagccaacaaggccacactggtgtgtctcataagtgactt region Constantctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtc Regionaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagt Nucleotideacgcggccagcagctacctgagcctgacgcctgagcagtggaagtccca Sequencecaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 231 Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV ChainKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEK Constant TVAPTECSRegion Amino Acid Sequence 232 Human Cλ IGCA3*04 Cλ Lightggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctg constant Chainaggagcttcaagccaacaaggccacactggtgtgtctcataagtgactt region Constantctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtc Regionaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagt Nucleotideacgcggccagcagctacctgagcctgacgcctgagcagtggaagtccca Sequencecagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 233 Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV ChainKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK Constant TVAPTECSRegion Amino Acid Sequence 234 Human Cλ IGCA6*01 Cλ Lightggtcagcccaaggctgccccatcggtcactctgttcccgccctcctctg constant Chainaggagcttcaagccaacaaggccacactggtgtgcctgatcagtgactt region Constantctacccgggagctgtgaaagtggcctggaaggcagatggcagccccgtc Regionaacacgggagtggagaccaccacaccctccaaacagagcaacaacaagt Nucleotideacgcggccagcagctacctgagcctgacgcctgagcagtggaagtccca Sequencecagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgttca 235 Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPV ChainNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK Constant TVAPAECSRegion Amino Acid Sequence 236 Human Cλ IGLC7*01 Cλ Lightggtcagcccaaggctgccccatcggtcactctgttcccaccctcctctg constant & Chainaggagcttcaagccaacaaggccacactggtgtgtctcgtaagtgactt region IGCA7*02Constant ctacccgggagccgtgacagtggcctggaaggcagatggcagccccgtc Regionaaggtgggagtggagaccaccaaaccctccaaacaaagcaacaacaagt Nucleotideatgcggccagcagctacctgagcctgacgcccgagcagtggaagtccca Sequencecagaagctacagctgccgggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgctct 237 Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV ChainKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEK Constant TVAPAECSRegion Amino Acid Sequence 238 413G05- Amino acid sequence GFTFSDYYCDRH1 of CDRH1 of 413G05 (IMGT) using IMGT 239 413G05-Amino acid sequence ISTSGSTI CDRH2 of CDRH2 of 413G05 (IMGT) using IMGT240 413G05- Amino acid sequence ARGITGTNFYHYGLGV CDRH3of CDRH3 of 413G05 (IMGT) using IMGT 241 413G05- Amino acid sequenceDYYMS CDRH1 of CDRH1 of 413G05 (Kabat) using Kabat 242 413G05-Amino acid sequence YISTSGSTIYYADSVKG CDRH2 of CDRH2 of 413G05 (Kabat)using Kabat 243 413G05- Amino acid sequence GITGTNFYHYGLGV CDRH3of CDRH3 of 413G05 (Kabat) using Kabat 244 413G05- Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQVPGKGLEWVS Heavyof V_(H) of 413G05 YISTSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDAAVYHCARchain GITGTNFYHYGLGVWGQGTTVTVSS variable region 245 413G05- Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT Heavysequence of V_(H) of CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTAchain 413G05 CATGAGCTGGATCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTTCA variableTACATTAGTACTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG regionGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACGCGGCCGTGTATCACTGTGCGAGAGGTATAACTGGAACTAACTTCTACCACTACGGTTTGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG 246 413G05- Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQVPGKGLEWVS full of 413G05 heavyYISTSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDAAVYHCAR heavy chainGITGTNFYHYGLGVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA chainLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS sequenceSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 247413G05- Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTfull sequence of 413G05CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA heavy heavy chainCATGAGCTGGATCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTTCA chainTACATTAGTACTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG sequenceGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACGCGGCCGTGTATCACTGTGCGAGAGGTATAACTGGAACTAACTTCTACCACTACGGTTTGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 248 413G05- Amino acid sequenceQGINSW CDRL1 of CDRL1 of 413G05 (IMGT) using IMGT 249 413G05-Amino acid sequence AAS CDRL2 of CDRL2 of 413G05 (IMGT) using IMGT 250413G05- Amino acid sequence QQVNSFPLT CDRL3 of CDRL3 of 413G05 (IMGT)using IMGT 251 413G05- Amino acid sequence RASQGINSWLA CDRL1of CDRL1 of 413G05 (Kabat) using Kabat 252 413G05- Amino acid sequenceAASTLQS CDRL2 of CDRL2 of 413G05 (Kabat) using Kabat 253 413G05-Amino acid sequence QQVNSFPLT CDRL3 of CDRL3 of 413G05 (Kabat)using Kabat 254 413G05- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPKLLIY Lightof V_(L) of 413G05 AASTLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQVNSFPLTFchain GGGTKVEIK variable region 255 413G05- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG Lightsequence of V_(L) of ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAACAGCTGGTTchain 413G05 AGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT variableGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG regionGGTCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC 256 413G05- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPKLLIY full of 413G05AASTLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQVNSFPLTF light chainGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 257 413G05- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG fullsequence of 413G05 ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAACAGCTGGTTlight light chain AGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATchain GCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG sequenceGGTCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 258 413F09-Amino acid sequence GFTFSYYA CDRH1 of CDRH1 of 413F09 (IMGT) using IMGT259 413F09- Amino acid sequence ISGGGGNT CDRH2 of CDRH2 of 413F09 (IMGT)using IMGT 260 413F09- Amino acid sequence AKDRMKQLVRAYYFDY CDRH3of CDRH3 of 413F09 (IMGT) using IMGT 261 413F09- Amino acid sequenceYYAMS CDRH1 of CDRH1 of 413F09 (Kabat) using Kabat 262 413F09-Amino acid sequence TISGGGGNTHYADSVKG CDRH2 of CDRH2 of 413F09 (Kabat)using Kabat 263 413F09- Amino acid sequence DRMKQLVRAYYFDY CDRH3of CDRH3 of 413F09 (Kabat) using Kabat 264 413F09- Amino acid sequenceEVPLVESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLDWVS Heavyof V_(H) of 413F09 TISGGGGNTHYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCAKchain DRMKQLVRAYYFDYWGQGTLVTVSS variable region 265 413F09- Nucleic acidGAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGT Heavysequence of V_(H) of CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCTACTATGCchain 413F09 CATGAGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGACTGGGTCTCA variableACTATTAGTGGTGGTGGTGGTAACACACACTACGCAGACTCCGTGAAGG regionGCCGATTCACTATATCCAGAGACAATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAAGACACGGCCGTCTATTACTGTGCGAAGGATCGGATGAAACAGCTCGTCCGGGCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG 266 413F09- Amino acid sequenceEVPLVESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLDWVS full of 413F09 heavyTISGGGGNTHYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCAK heavy chainDRMKQLVRAYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA chainLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS sequenceSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 267413F09- Nucleic acid GAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTfull sequence of 413F09CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCTACTATGC heavy heavy chainCATGAGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGACTGGGTCTCA chainACTATTAGTGGTGGTGGTGGTAACACACACTACGCAGACTCCGTGAAGG sequenceGCCGATTCACTATATCCAGAGACAATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAAGACACGGCCGTCTATTACTGTGCGAAGGATCGGATGAAACAGCTCGTCCGGGCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 268 413F09- Amino acid sequenceQDISTY CDRL1 of CDRL1 of 413F09 (IMGT) using IMGT 269 413F09-Amino acid sequence GTS CDRL2 of CDRL2 of 413F09 (IMGT) using IMGT 270413F09- Amino acid sequence QQLHTDPIT CDRL3 of CDRL3 of 413F09 (IMGT)using IMGT 271 413F09- Amino acid sequence WASQDISTYLG CDRL1of CDRL1 of 413F09 (Kabat) using Kabat 272 413F09- Amino acid sequenceGTSSLQS CDRL2 of CDRL2 of 413F09 (Kabat) using Kabat 273 413F09-Amino acid sequence QQLHTDPIT CDRL3 of CDRL3 of 413F09 (Kabat)using Kabat 274 413F09- Amino acid sequenceDIQLTQSPSFLSASVGDRVTITCWASQDISTYLGWYQQKPGKAPKLLIY Light of VL of 413F09GTSSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLHTDPITF chain GQGTRLEIKvariable region 275 413F09- Nucleic acidGACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAG Lightsequence of VL of ACAGAGTCACCATCACTTGCTGGGCCAGTCAGGACATTAGCACTTATTTchain 413F09 AGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT variableGGTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG regionGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTCATACTGACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC 276 413F09- Amino acid sequenceDIQLTQSPSFLSASVGDRVTITCWASQDISTYLGWYQQKPGKAPKLLIY full of 413F09 lightGTSSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLHTDPITF light chainGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 277 413F09- Nucleic acidGACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAG fullsequence of 413F09 ACAGAGTCACCATCACTTGCTGGGCCAGTCAGGACATTAGCACTTATTTlight light chain AGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATchain GGTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG sequenceGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTCATACTGACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 278 414B06-Amino acid sequence GFTFSSYW CDRH1 of CDRH1 of 414B06 (IMGT) using IMGT279 414B06- Amino acid sequence IKQDGSEK CDRH2 of CDRH2 of 414B06 (IMGT)using IMGT 280 414B06- Amino acid sequence ARVRQWSDYSDY CDRH3of CDRH3 of 414B06 (IMGT) using IMGT 281 414B06- Amino acid sequenceSYWMN CDRH1 of CDRH1 of 414B06 (Kabat) using Kabat 282 414B06-Amino acid sequence NIKQDGSEKYYVDSVKG CDRH2 of CDRH2 of 414B06 (Kabat)using Kabat 283 414B06- Amino acid sequence VRQWSDYSDY CDRH3of CDRH3 of 414B06 (Kabat) using Kabat 284 414B06- Amino acid sequenceEVHLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVA Heavy of VH of 414B06NIKQDGSEKYYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCAR chainVRQWSDYSDYWGQGTPVTVSS variable region 285 414B06- Nucleic acidGAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT Heavysequence of VH of CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGchain 414B06 GATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCC variableAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGG regionGCCGCTTCACCGTCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTCGACAATGGTCCGACTACTCTGACTACTGGGGCCAGGGAACCCCGG TCACCGTCTCCTCAG 286414B06- Amino acid sequenceEVHLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVA full of 414B06 heavyNIKQDGSEKYYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCAR heavy chainVRQWSDYSDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL chainVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG sequenceTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 287 414B06-Nucleic acid GAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGT fullsequence of 414B06 CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGheavy heavy chain GATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCchain AACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGG sequenceGCCGCTTCACCGTCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTCGACAATGGTCCGACTACTCTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 288 414B06- Amino acid sequence QGISSW CDRL1of CDRL1 of 414B06 (IMGT) using IMGT 289 414B06- Amino acid sequence AASCDRL2 of CDRL2 of 414B06 (IMGT) using IMGT 290 414B06-Amino acid sequence QQANSFPFT CDRL3 of CDRL3 of 414B06 (IMGT) using IMGT291 414B06- Amino acid sequence RASQGISSWLA CDRL1 of CDRL1 of 414B06(Kabat) using Kabat 292 414B06- Amino acid sequence AASSLQS CDRL2of CDRL2 of 414B06 (Kabat) using Kabat 293 414B06- Amino acid sequenceQQANSFPFT CDRL3 of CDRL3 of 414B06 (Kabat) using Kabat 294 414B06-Amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYLight of VL of 414B06 AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFchain GPGTKVDIK variable region 295 414B06- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG Lightsequence of VL of ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTchain 414B06 AGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT variableGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG regionGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 296 414B06- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIY full of 414B06 lightAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTF light chainGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequenceTHQGLSSPVTKSFNRGEC 297 414B06- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG fullsequence of 414B06 ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTlight light chain AGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATchain GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG sequenceGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCG AGTGT 298 MutatedAmino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY1D05-LC of 1D05 kappa light YASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITF mutant 3chain with V to Y GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ mutation in CDRL2 WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVhighlighted THQGLSSPVTKSFNRGEC 299 1D05- Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS heavy of IgG1 disabledGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCAK chain variant of 1D05DMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA disabledLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS IgG1 FcSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 300 1D05-1D05 Light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY lightsequence fused to VASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFchain IL- wild-type human IL-2GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ  2 fusionsequence (IL-2 amino WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVacid sequence is THQGLSSPVTKSFNRGEC APTSSSTKKTQLQLEHLLLDLQMILNGINNYunderlined and KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLregion to be varied RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIis shown in bold) STLT 301 Human IL-2 Uniprot number:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK P60568ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKG Full length aminoSETTFMCEYADETATIVEFLNRWITFCQSIISTLT acid sequence of human IL-2 (minussignal sequence) 302 Control Heavy chain 1D05EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS 1D05IgG1 variant fused GISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCAKimmunocyt at the N-terminus toDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA okine HCwild-type human IL2 LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS C-sequence (control) SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LAGA PSterminal VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK fusionTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 303 IL-2 D5-9IL-2 1045 (Del 5-9) APTSTQLQLELLLD N terminal IL-2 sequence 304IL-2 D1-9 IL-2 1046 (Del 1-9) TQLQLEHLLLD N terminal IL-2 sequence 305IL-2 D5-7 IL-2 1064 (Del 5-7) APTSKKTQLQLEHLLLD N terminal IL-2 sequence306 IL-2 D1 IL-2 D1 N terminal PTSSSTKKTQLQLEHLLLD IL-2 sequence 307IL-2 D1-2 IL-2 D1-2 N terminal TSSSTKKTQLQLEHLLLD IL-2 sequence 308IL-2 D1-3 IL-2 D1-3 N terminal SSSTKKTQLQLEHLLLD IL-2 sequence 309IL-2 D1-4 IL-2 D1-4 N terminal SSTKKTQLQLEHLLLD IL-2 sequence 310IL-2 D1-5 IL-2 D1-5 N terminal STKKTQLQLEHLLLD IL-2 sequence 311IL-2 D1-6 IL-2 D1-6 N terminal TKKTQLQLEHLLLD IL-2 sequence 312IL-2 D1-7 IL-2 D1-7 N terminal KKTQLQLEHLLLD IL-2 sequence 313 IL-2 D1-8IL-2 D1-8 N terminal KTQLQLEHLLLD IL-2 sequence 314 IL-2 D9IL-2 D9 N terminal APTSSSTKTQLQLEHLLLD IL-2 sequence 315 IL-2 D9-8IL-2 D9-8 N terminal APTSSSTTQLQLEHLLLD IL-2 sequence 316 IL-2 D9-7IL-2 D9-7 N terminal APTSSSTQLQLEHLLLD IL-2 sequence 317 IL-2 D9-6IL-2 D9-6 N terminal APTSSTQLQLEHLLLD IL-2 sequence 318 IL-2 D9-4IL-2 D9-4 N terminal APTTQLQLEHLLLD IL-2 sequence 319 IL-2 D9-3IL-2 D9-3 N terminal APTQLQLEHLLLD IL-2 sequence 320 IL-2 D9-2IL-2 D9-2 N terminal ATQLQLEHLLLD IL-2 sequence 321 IL-2 D2-6IL-2 D2-6 N terminal ATKKTQLQLEHLLLD IL-2 sequence 322 IL-2 D3-7IL-2 D3-7 N terminal APKKTQLQLEHLLLD IL-2 sequence 323 IL-2 D4-8IL-2 D4-8 N terminal APTKTQLQLEHLLLD IL-2 sequence 324 C-Amino acids 21 to LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVterminal 133 of hIL-2 LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLamino NRWITFCQSIISTLT acid sequence of hIL-2 325 Mouse PD-Uniprot number: MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFPVERELD L1Q9EP73 LLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAA(ECD highlighted in LQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATBOLD, and SEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLcytoplasmic domain RVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRT HWVLLGSunderlined) ILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET 326 Mouse PD-Mouse PD-L1 FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQF L1 ECDextracellular domain VAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIIHis  with his tag SYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQ PGQNHTAELIIPELPATHPPQNRT HHHHHH 327 Human IL- Human IL-2 receptorELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGN 2Rα chain alpha chainSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQR KSRRTI 328 Human IL-Human IL-2 receptor AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCE2Rβ chain beta chain LLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV 329 Human IL- Human IL-2 receptorLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMN 2Rγ chaincommon gamma chain CTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQ LELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTL KPET 330 IL-7Human IL-7 amino DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAacid sequence NKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILM GTKEH 331 IL-15Human IL-15 amino GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDacid sequence VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 332 IL-21 Human IL-21 aminoQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSC acid sequenceFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS 333 GM-CSF Human GM-CSF aminoAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDL acid sequenceQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE 334 IFNα Human IFN-a aminoCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQ  acid sequenceKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTELHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGS 335 TNFα ExtracellularGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWL portion of humanNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTH TNF-α amino acidTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLE sequenceKGDRLSAEINRPDYLDFAESGQVYFGIIAL 336 IL-12α Alpha chain of humanRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH IL-12 amino acidEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM sequenceALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNA S 337 IL-12βBeta chain of human IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSIL-12 amino acid GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQ sequence KEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYS SSWSEWASVPCS 338 CXCL9Human CXCL-9 amino TPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVacid sequence QTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSR QKKTT339 CXCL10 Human CXCL-10 aminoVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKG acid sequenceEKRCLNPESKAIKNLLKAVSKERSKRSP 340 Human WT IGHG1*01  WT humanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG IgG1 & IgG1 aminoVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV constant IGHG1*02 acidEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV region & sequenceDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW IGHG1*05LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ  (IgG1)VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 341 WT humanGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGT IgG1CCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTT nucleicCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGA acidGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGT sequenceCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAG CCCCGGCAAGTGATGA 342Mutated Amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ APGKGLEWVS 1D05-HC of 1D05 heavy chain GISWIRTGIGYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAK mutant 2 with V to A and F toDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA S back-mutation inLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS framework region toSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE LAGA PSVFL germline highlightedFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP with IgG1 disabledREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (LAGA) constantGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN regionNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ  KSLSLSLGK

TABLE S2 SEQ ID NOS: 343-538 SEQ ID NO: Name Description Sequence 343416E01- Amino acid sequence GFTFSNYA CDRH1 of CDRH1 of 416E01 (IMGT)using IMGT 344 416E01- Amino acid sequence ISFSGGTT CDRH2of CDRH2 of 416E01 (IMGT) using IMGT 345 416E01- Amino acid sequenceAKDEAPAGATFFDS CDRH3 of CDRH3 of 416E01 (IMGT) using IMGT 346 416E01-Amino acid sequence NYAMS CDRH1 of CDRH1 of 416E01 (Kabat) using Kabat347 416E01- Amino acid sequence AISFSGGTTYYADSVKG CDRH2of CDRH2 of 416E01 (Kabat) using Kabat 348 416E01- Amino acid sequenceDEAPAGATFFDS CDRH3 of CDRH3 of 416E01 (Kabat) using Kabat 349 416E01-Amino acid sequence EVQLAESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQTPGKGLEWHeavy of V_(H) of 416E01 VSAISFSGGTTYYADSVKGRFTISRDNSKNTLYLHMNSLRADDTAVYchain (mutations from YCAKDEAPAGATFFDSWGQGTLVTVSS variablegermline are shown region in bold letters) 350 416E01- Nucleic acidGAAGTGCAACTGGCGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGG Heavysequence of V_(H) of GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAACTchain 416E01 ATGCCATGAGTTGGGTCCGCCAGACTCCAGGAAAGGGGCTGGAGTGG variableGTCTCAGCTATTAGTTTTAGTGGTGGTACTACATACTACGCTGACTC regionCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCACATGAACAGCCTGAGAGCCGATGACACGGCCGTATATTACTGTGCGAAAGATGAGGCACCAGCTGGCGCAACCTTCTTTGACTCCTGGGGCCAGGGAACGCTGGTCACCGTCTCCTCAG 351 416E01- Amino acid sequenceEVQLAESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQTPGKGLEW full of 416E01 heavyVSAISFSGGTTYYADSVKGRFTISRDNSKNTLYLHMNSLRADDTAVY heavy chainYCAKDEAPAGATFFDSWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE chainSTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS sequenceVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 352 416E01- Nucleic acidGAAGTGCAACTGGCGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGG full sequence of 416E01GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAACT heavy heavy chainATGCCATGAGTTGGGTCCGCCAGACTCCAGGAAAGGGGCTGGAGTGG chainGTCTCAGCTATTAGTTTTAGTGGTGGTACTACATACTACGCTGACTC sequenceCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCACATGAACAGCCTGAGAGCCGATGACACGGCCGTATATTACTGTGCGAAAGATGAGGCACCAGCTGGCGCAACCTTCTTTGACTCGCCCTTCCGTGTTCCCCCTGGCCCCTTGCAGCAGGAGCACCTCCGAATCCACAGCTGCCCTGGGCTGTCTGGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCTCTGACATCCGGCGTCCACACCTTTCCTGCCGTCCTGCAGTCCTCCGGCCTCTACTCCCTGTCCTCCGTGGTGACCGTGCCTAGCTCCTCCCTCGGCACCAAGACCTACACCTGTAACGTGGACCACAAACCCTCCAACACCAAGGTGGACAAACGGGTCGAGAGCAAGTACGGCCCTCCCTGCCCTCCTTGTCCTGCCCCCGAGTTCGAAGGCGGACCCAGCGTGTTCCTGTTCCCTCCTAAGCCCAAGGACACCCTCATGATCAGCCGGACACCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTATGTGGATGGCGTGGAGGTGCACAACGCCAAGACAAAGCCCCGGGAAGAGCAGTTCAACTCCACCTACAGGGTGGTCAGCGTGCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCAGCAATAAGGGACTGCCCAGCAGCATCGAGAAGACCATCTCCAAGGCTAAAGGCCAGCCCCGGGAACCTCAGGTGTACACCCTGCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCTCCCGTCCTCGACAGCGACGGATCCTTCTTTCTGTACTCCAGGCTGACCGTGGATAAGTCCAGGTGGCAGGAAGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGAGCCTGTCCCTGGGAAAG 353 416E01- Amino acid sequence QGIRRWCDRL1 of CDRL1 of 416E01 (IMGT) using IMGT 354 416E01-Amino acid sequence GAS CDRL2 of CDRL2 of 416E01 (IMGT) using IMGT 355416E01- Amino acid sequence QQANSFPIT CDRL3 of CDRL3 of 416E01 (IMGT)using IMGT 356 416E01- Amino acid sequence RASQGIRRWLA CDRL1of CDRL1 of 416E01 (Kabat) using Kabat 357 416E01- Amino acid sequenceGASSLQS CDRL2 of CDRL2 of 416E01 (Kabat) using Kabat 358 416E01-Amino acid sequence QQANSFPIT CDRL3 of CDRL3 of 416E01 (Kabat)using Kabat 359 416E01- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIRRWLAWYQQKPGKAPKLL Light of V_(L) ofISGASSLQSGVPSRFSGSGSGTDFTLIITSLQPEDFATYYCQQANSF chain 416E01 (mutationsPITFGQGTRLEIK variable from germline are region shown in bold letters)360 416E01- Nucleic acid GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGLight sequence of V_(L) ofAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGGAGGT chain 416E01GGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTG variableATCTCTGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAG regionCGGCAGTGGATCTGGGACAGATTTCACTCTCATCATTACCAGTCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC 361 416E01- Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIRRWLAWYQQKPGKAPKLL full of 416E01 lightISGASSLQSGVPSRFSGSGSGTDFTLIITSLQPEDFATYYCQQANSF light chainPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP chainREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK sequenceHKVYACEVTHQGLSSPVTKSFNRGEC 362 416E01- Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGG full sequence of 416E01AGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGGAGGT light light chainGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTG chainATCTCTGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAG sequenceCGGCAGTGGATCTGGGACAGATTTCACTCTCATCATTACCAGTCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 363 STIM001- Amino acid sequenceGYTFSTFG CDRH1 of CDRH1 of STIM001 using IMGT 364 STIM001-Amino acid sequence ISAYNGDT CDRH2 of CDRH2 of STIM001 using IMGT 365STIM001- Amino acid sequence ARSSGHYYYYGMDV CDRH3 of CDRH3 of STIM001using IMGT 366 STIM001- Amino acid sequenceQVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEW Heavyof V_(H) of STIM001 MGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAVYchain YCARSSGHYYYYGMDVWGQGTTVTVSS variable region 367 STIM001-Nucleic acid CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGC Heavysequence of V_(H) of CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACCTchain STIM001 TTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGG variableATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACAGAA regionTCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 368 STIM001- Amino acid sequenceQVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEW full of STIM001 heavyMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAVY heavy chainYCARSSGHYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG chainGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS sequenceVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 369 STIM001- Nucleic acidCAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGC full sequence of STIM001CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACCT heavy heavy chainTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGG chainATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACAGAA sequenceTCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA 370 STIM001-Amino acid sequence QSLLHSNEYNY CDRL1 of CDRL1 of STIM001 using IMGT 371STIM001- Amino acid sequence LGS CDRL2 of CDRL2 of STIM001 using IMGT372 STIM001- Amino acid sequence MQSLQTPLT CDRL3 of CDRL3 of STIM001using IMGT 373 STIM001- Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGQ Lightof V_(L) of STIM001 SPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYCMchain QSLQTPLTFGGGTKVEIK variable region 374 STIM001- Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG Lightsequence of V_(L) of AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAchain STIM001 GTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAG variableTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGGGGT regionCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGA GATCAA 375 STIM001-Amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGQ fullof STIM001 light SPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYCM lightchain QSLQTPLTFGGGTKVEIK chainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL sequenceQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 376STIM001- Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGfull sequence of STIM001 AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAlight light chain GTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAG chainTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGGGGT sequenceCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 377 STIM002-Amino acid sequence GYTFTSYG CDRH1 of CDRH1 of STIM002 using IMGT 378STIM002- Amino acid sequence ISAYNGNT CDRH2 of CDRH2 of STIM002using IMGT 379 STIM002- Amino acid sequence ARSTYFYGSGTLYGMDV CDRH3of CDRH3 of STIM002 using IMGT 380 STIM002- Amino acid sequenceQVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEW Heavyof V_(H) of STIM002 MGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYchain YCARSTYFYGSGTLYGMDVWGQGTTVTVSS variable region 381 STIM002-Nucleic acid CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGC Heavysequence of V_(H) of CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTchain STIM002 ATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGG variableATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAA regionGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 382 STIM002-Amino acid sequence QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEW fullof STIM002 heavy MGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVY heavychain YCARSTYFYGSGTLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383 STIM002- Nucleic acidCAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGC full sequence of STIM002CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCT heavy heavy chainATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGG chainATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAA sequenceGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGC AAGTGATGA 384 STIM002-Amino acid sequence QSLLHSDGYNY CDRL1 of CDRL1 of STIM002 using IMGT 385STIM002- Amino acid sequence LGS CDRL2 of CDRL2 of STIM002 using IMGT386 STIM002- Amino acid sequence MQALQTPLS CDRL3 of CDRL3 of STIM002using IMGT 387 STIM002- Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQ Lightof V_(L) of STIM002 SPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMchain QALQTPLSFGQGTKLEIK variable region 388 STIM002- Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG Lightsequence of V_(L) of AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAchain STIM002 GTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAG variableTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTT regionCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGA GATCAA 389 STIM002-Amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQ fullof STIM002 light SPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM lightchain QALQTPLSFGQGTKLEIK chainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL sequenceQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 390STIM002- Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGfull sequence of STIM002 AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAlight light chain GTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAG chainTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTT sequenceCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 391 STIM002-B-Amino acid sequence GYTFTSYG CDRH1 of CDRH1 of STIM002- B using IMGT 392STIM002-B- Amino acid sequence ISAYNGNT CDRH2 of CDRH2 of STIM002-B using IMGT 393 STIM002-B- Amino acid sequence ARSTYFYGSGTLYGMDV CDRH3of CDRH3 of STIM002- B using IMGT 394 STIM002-B- Amino acid sequenceQVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEW Heavyof V_(H) of STIM002-B MGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYchain YCARSTYFYGSGTLYGMDVWGQGTTVTVSS variable region 395 STIM002-B-Nucleic acid CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGC Heavysequence of V_(H) of CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTchain STIM002-B ATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGG variableATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAA regionGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 396 STIM002-B-Amino acid sequence QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEW fullof STIM002-B heavy MGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVY heavychain YCARSTYFYGSGTLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 397 STIM002-B- Nucleic acidCAGGTTCAACTGCTGCACTCTGGAGCTGAGCTGAAGAAGCCTCGGGC fullsequence of STIM002- CTCAGTGAAGGTCTCCTGCAAGGCTTCTGCTTACACCTTTACCAGCTheavy B heavy chain ATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGchain ATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAA sequenceGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGAGCGTGGCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGT GATGA 398 STIM002-B-Amino acid sequence QSLLHSDGYNC CDRL1 of CDRL1 of STIM002- B using IMGT399 STIM002-B- Amino acid sequence LGS CDRL2 of CDRL2 of STIM002-B using IMGT 400 STIM002-B- Amino acid sequence MQALQTPCS CDRL3of CDRL3 of STIM002- B using IMGT 401 STIM002-B- Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGQ Lightof V_(L) of STIM002-B SPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMchain QALQTPCSFGQGTKLEIK variable region 402 STIM002-B- Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG Lightsequence of V_(L) of AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAchain STIM002-B GTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAG variableTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTT regionCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGA GATCAA 403 STIM002-B-Amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGQ fullof STIM002-B light SPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM lightchain QALQTPCSFGQGTKLEIK chainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL sequenceQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 404STIM002-B- Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGfull sequence of STIM002-AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATA light B light chainGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAG chainTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTT sequenceCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 405 STIM003-Amino acid sequence GVTFDDYG CDRH1 of CDRH1 of STIM003 using IMGT 406STIM003- Amino acid sequence INWNGGDT CDRH2 of CDRH2 of STIM003using IMGT 407 STIM003- Amino acid sequence ARDFYGSGSYYHVPFDY CDRH3of CDRH3 of STIM003 using IMGT 408 STIM003- Amino acid sequenceEVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEW Heavyof V_(H) of STIM003 VSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYchain YCARDFYGSGSYYHVPFDYWGQGILVTVSS variable region 409 STIM003-Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGG Heavysequence of V_(H) of GTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTchain STIM003 ATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARTGG variableGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTC regionTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA 410 STIM003-Amino acid sequence EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEW fullof STIM003 heavy VSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALY heavychain YCARDFYGSGSYYHVPFDYWGQGILVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 411 STIM003- Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGG full sequence of STIM003GTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATT heavy heavy chainATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARTGG chainGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTC sequenceTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGT GATGA 412 STIM003-Amino acid sequence QSVSRSY CDRL1 of CDRL1 of STIM003 using IMGT 413STIM003- Amino acid sequence GAS CDRL2 of CDRL2 of STIM003 using IMGT414 STIM003- Amino acid sequence HQYDMSPFT CDRL3 of CDRL3 of STIM003using IMGT 415 STIM003- Amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRL Lightof V_(L) of STIM003 LIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYDMchain SPFTFGPGTKVDIK variable region 416 STIM003- Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGG Lightsequence of V_(L) of GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGAAchain STIM003 GCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGCTC variableCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTT regionCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA 417 STIM003-Amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRL fullof STIM003 light LIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYDM lightchain SPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY chainPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE sequenceKHKVYACEVTHQGLSSPVTKSFNRGEC 418 STIM003- Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGG full sequence of STIM003GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGAA light light chainGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGCTC chainCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTT sequenceCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 419 STIM004- Amino acid sequenceGLTFDDYG CDRH1 of CDRH1 of STIM004 using IMGT 420 STIM004-Amino acid sequence INWNGDNT CDRH2 of CDRH2 of STIM004 using IMGT 421STIM004- Amino acid sequence ARDYYGSGSYYNVPFDY CDRH3 of CDRH3 of STIM004using IMGT 422 STIM004- Amino acid sequenceEVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEW Heavyof V_(H) of STIM004 VSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYchain YCARDYYGSGSYYNVPFDYWGQGTLVTVSS variable region 423 STIM004-Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGG Heavysequence of V_(H) of GTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGATTchain STIM004 ATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGG variableGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGACTC regionTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 424 STIM004-Amino acid sequence EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEW fullof STIM004 heavy VSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALY heavychain YCARDYYGSGSYYNVPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 425 STIM004- Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGG full sequence of STIM004GTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGATT heavy heavy chainATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGG chainGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGACTC sequenceTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTCACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGT GATGA 426 STIM004-Amino acid sequence QSVSSSY CDRL1 of CDRL1 of STIM004 using IMGT 427STIM004- Amino acid sequence GAS CDRL2 of CDRL2 of STIM004 using IMGT428 STIM004- Amino acid sequence QQYGSSPF CDRL3 of CDRL3 of STIM004using IMGT 429 STIM004- Amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL Correctedof corrected V_(L) of LIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYGSlight STIM004 SPFFGPGTKVDIK chain variable region 430 STIM004-Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGG Correctedsequence of GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCA lightcorrected V_(L) of GCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTC chainSTIM004 CTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTT variableCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGAC regionTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAA 431 STIM004- Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGG Lightsequence of V_(L) of GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAchain STIM004 GCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTC variableCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTT regionCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAA 432 STIM004-Amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL fullof STIM004 light LIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYGScorrected chain SPFFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP lightREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK chainHKVYACEVTHQGLSSPVTKSFNRGEC sequence 433 STIM004- Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGG full sequence ofGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCA correctedcorrected STIM004 GCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTC lightlight chain CTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTT chainCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGAC sequenceTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 434 STIM004- Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGG full sequence of STIM004GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCA light light chainGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTC chainCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTT sequenceCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 435 STIM005- Amino acid sequenceGYTFNSYG CDRH1 of CDRH1 of STIM005 using IMGT 436 STIM005-Amino acid sequence ISVHNGNT CDRH2 of CDRH2 of STIM005 using IMGT 437STIM005- Amino acid sequence ARAGYDILTDFSDAFDI CDRH3 of CDRH3 of STIM005using IMGT 438 STIM005- Amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEW Heavyof V_(H) of STIM005 MGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAVYchain YCARAGYDILTDFSDAFDIWGHGTMVTVSS variable region 439 STIM005-Nucleic acid CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGC Heavysequence of V_(H) of CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGTTchain STIM005 ATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGG variableATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACAGAA regionGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCA 440 STIM005-Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEW fullof STIM005 heavy MGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAVY heavychain YCARAGYDILTDFSDAFDIWGHGTMVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 441 STIM005- Nucleic acidCAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGC full sequence of STIM005CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGTT heavy heavy chainATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGG chainATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACAGAA sequenceGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGC AAGTGATGA 442 STIM005-Amino acid sequence QNINNF CDRL1 of CDRL1 of STIM005 using IMGT 443STIM005- Amino acid sequence AAS CDRL2 of CDRL2 of STIM005 using IMGT444 STIM005- Amino acid sequence QQSYGIPW CDRL3 of CDRL3 of STIM005using IMGT 445 STIM005- Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLL Lightof V_(L) of STIM005 IYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYGIchain PWVGQGTKVEIK variable region 446 STIM005- Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG Lightsequence of V_(L) of AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAACTchain STIM005 TTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCCTG variableATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTTCAG regionTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAA 447 STIM005- Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLL full of STIM005 lightIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYGI light chain PWVGQGTKVEIKchain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL sequenceQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 448STIM005- Nucleic acid GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGfull sequence of STIM005 AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAACTlight light chain TTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCCTG chainATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTTCAG sequenceTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 449 STIM006- Amino acid sequence GFTFSDYFCDRH1 of CDRH1 of STIM006 using IMGT 450 STIM006- Amino acid sequenceISSSGSTI CDRH2 of CDRH2 of STIM006 using IMGT 451 STIM006-Amino acid sequence ARDHYDGSGIYPLYYYYGLDV CDRH3 of CDRH3 of STIM006using IMGT 452 STIM006- Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEW Heavyof V_(H) of STIM006 ISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAVYchain YCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSS variable region 453 STIM006-Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGG Heavysequence of V_(H) of GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTchain STIM006 ACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGTGG variableATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGACTC regionTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCG TCTCCTCA 454 STIM006-Amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEW fullof STIM006 heavy ISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAVY heavychain YCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSSASTKGPSVFPLAP chainSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS sequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 455 STIM006- Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGG full sequence of STIM006GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACT heavy heavy chainACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGTGG chainATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGACTC sequenceTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGA GCCCCGGCAAGTGATGA 456STIM006- Amino acid sequence QSLLHSNGYNY CDRL1 of CDRL1 of STIM006using IMGT 457 STIM006- Amino acid sequence LGS CDRL2of CDRL2 of STIM006 using IMGT 458 STIM006- Amino acid sequenceMQALQTPRS CDRL3 of CDRL3 of STIM006 using IMGT 459 STIM006-Amino acid sequence IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQSLight of V_(L) of STIM006PQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ chain ALQTPRSFGQGTTLEIKvariable region 460 STIM006- Nucleic acidATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGA Lightsequence of V_(L) of GCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAchain STIM006 ATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGTCT variableCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGTCCC regionTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGCTGGAGAT CAAA 461 STIM006-Amino acid sequence IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQS fullof STIM006 light PQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ lightchain ALQTPRSFGQGTTLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN chainNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequenceDYEKHKVYACEVTHQGLSSPVTKSFNRGEC 462 STIM006- Nucleic acidATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGA full sequence of STIM006GCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTA light light chainATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGTCT chainCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGTCCC sequenceTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGOTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 463 STIM007-Amino acid sequence GFSLSTTGVG CDRH1 of CDRH1 of STIM007 using IMGT 464STIM007- Amino acid sequence IYWDDDK CDRH2 of CDRH2 of STIM007using IMGT 465 STIM007- Amino acid sequence THGYGSASYYHYGMDV CDRH3of CDRH3 of STIM007 using IMGT 466 STIM007- Amino acid sequenceQITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKAL Heavyof V_(H) of STIM007 EWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATchain YFCTHGYGSASYYHYGMDVWGQGTTVTVSS variable region 467 STIM007-Nucleic acid CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACA Heavysequence of V_(H) of GACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAchain STIM007 CTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTG variableGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCC regionATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 468 STIM007-Amino acid sequence QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKAL fullof STIM007 heavy EWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTAT heavychain YFCTHGYGSASYYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 469 STIM007- Nucleic acidCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACA full sequence of STIM007GACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTA heavy heavy chainCTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTG chainGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCC sequenceATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGC AAGTGATGA 470 STIM007-Amino acid sequence QSVTNY CDRL1 of CDRL1 of STIM007 using IMGT 471STIM007- Amino acid sequence DAS CDRL2 of CDRL2 of STIM007 using IMGT472 STIM007- Amino acid sequence QHRSNWPLT CDRL3 of CDRL3 of STIM007using IMGT 473 STIM007- Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLL Lightof V_(L) of STIM007 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWchain PLTFGGGTKVEIK variable region 474 STIM007- Nucleic acidGAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGG Lightsequence of V_(L) of GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTchain STIM007 ACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTC variableATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG regionTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC 475 STIM007-Amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLL fullof STIM007 light IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNW lightchain PLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP chainREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK sequenceHKVYACEVTHQGLSSPVTKSFNRGEC 476 STIM007- Nucleic acidGAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGG full sequence of STIM007GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACT light light chainACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTC chainATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG sequenceTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 477 STIM008- Amino acid sequenceGFSLSTSGVG CDRH1 of CDRH1 of STIM008 using IMGT 478 STIM008-Amino acid sequence IYWDDDK CDRH2 of CDRH2 of STIM008 using IMGT 479STIM008- Amino acid sequence THGYGSASYYHYGMDV CDRH3 of CDRH3 of STIM008using IMGT 480 STIM008- Amino acid sequenceQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKAL Heavyof V_(H) of STIM008 EWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATchain YFCTHGYGSASYYHYGMDVWGQGTTVTVSS variable region 481 STIM008-Nucleic acid CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACA Heavysequence of V_(H) of GACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAchain STIM008 GTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTG variableGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCC regionATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 482 STIM008-Amino acid sequence QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKAL fullof STIM008 heavy EWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTAT heavychain YFCTHGYGSASYYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKS chainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequenceLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 483 STIM008- Nucleic acidCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACA full sequence of STIM008GACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTA heavy heavy chainGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTG chainGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCC sequenceATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGAGCGTGGCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGrGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGT GATGA 484 STIM008-Amino acid sequence QSVTNY CDRL1 of CDRL1 of STIM008 using IMGT 485STIM008- Amino acid sequence DAS CDRL2 of CDRL2 of STIM008 using IMGT486 STIM008- Amino acid sequence QQRSNWPLT CDRL3 of CDRL3 of STIM008using IMGT 487 STIM008- Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLL Lightof V_(L) of STIM008 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWchain PLTFGGGTKVEIK variable region 488 STIM008- Nucleic acidGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGG Lightsequence of V_(L) of GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTchain STIM008 ACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTC variableATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG regionTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 489 STIM008- Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLL full of STIM008 lightIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW light chainPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP chainREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK sequenceHKVYACEVTHQGLSSPVTKSFNRGEC 490 STIM008- Nucleic acidGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGG full sequence of STIM008GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACT light light chainACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTC chainATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG sequenceTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 491 STIM009- Amino acid sequenceGFTFSDYY CDRH1 of CDRH1 of STIM009 using IMGT 492 STIM009-Amino acid sequence ISSSGSTI CDRH2 of CDRH2 of STIM009 using IMGT 493STIM009- Amino acid sequence ARDFYDILTDSPYFYYGVDV CDRH3of CDRH3 of STIM009 using IMGT 494 STIM009- Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEW Heavyof V_(H) of STIM009 VSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAVYchain YCARDFYDILTDSPYFYYGVDVWGQGTTVTVSS variable region 495 STIM009-Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGG Heavysequence of V_(H) of GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTchain STIM009 ACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG variableGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTC regionTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CCTCA 496 STIM009-Amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEW fullof STIM009 heavy VSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAVY heavychain YCARDFYDILTDSPYFYYGVDVWGQGTTVTVSSASTKGPSVFPLAPS chainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequenceLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 497 STIM009- Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGG full sequence of STIM009GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACT heavy heavy chainACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG chainGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTC sequenceTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCC CCGGCAAGTGATGA 498STIM009- Amino acid sequence QSLLHSNGYNY CDRL1 of CDRL1 of STIM009using IMGT 499 STIM009- Amino acid sequence LGS CDRL2of CDRL2 of STIM009 using IMGT 500 STIM009- Amino acid sequenceMQALQTPRT CDRL3 of CDRL3 of STIM009 using IMGT 501 STIM009-Amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQLight of V_(L) of STIM009SPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM chain QALQTPRTFGQGTKVEIKvariable region 502 STIM009- Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG Lightsequence of V_(L) of AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAchain STIM009 GTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAG variableTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGT regionCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGA AATCAA 503 STIM009-Amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQ fullof STIM009 light SPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM lightchain QALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL chainNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK sequenceADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 504 STIM009- Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG full sequence of STIM009AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATA light light chainGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAG chainTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGT sequenceCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 505 Human PD-Amino acid sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIL1 Flag of KYPROT286 withQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVY His FLAG tag in bold andRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEG (KYPROT286)underlined and YPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIhistidine tag in FYCTFRRLDPEENHTAELVIPELPLAHPPNERTIEGR DYKDDDDK HH boldHHHH 506 Mature Mature amino acidEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTK human sequence of humanTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIF ICOS ICOSDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL 507 Human Amino acid sequenceEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTK ICOS of human ICOSTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIF extracellularextracellular domain DPPPFKVTLTGGYLHIYESQLCCQLKF domain 508 HumanAmino acid sequence MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIICOS with of human ICOS VQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFsignal (signal peptide isFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKF peptide underlined)WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNT AKKSRLTDVTL 509 IsoformAmino acid sequence The sequence of this isoform differs from theof human of a human ICOS canonical sequence in its cytoplasmic domain asICOS isoform follows: 168-199: (Q9Y6W8-2)KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLM 510 Mature Mature amino acidEINGSADHRMFSFHNGGVQISCKYPETVQQLKMRLFREREVLCELTK mouse sequence of mouseTKGSGNAVSIKNPMLCLYHLSNNSVSFFLNNPDSSQGSYYFCSLSIF ICOS ICOSDPPPFQERNLSGGYLHIYESQLCCQLKIVVQVTE 511 Mouse Amino acid sequenceEINGSADHRMFSFHNGGVQISCKYPETVQQLKMRLFREREVLCELTK ICOSof the extracellular TKGSGNAVSIKNPMLCLYHLSNNSVSFFLNNPDSSQGSYYFCSLSIFextracellular domain of mouse ICOS DPPPFQERNLSGGYLHIYESQLCCQLK domain512 Mouse Amino acid sequenceMGWSCIILFLVATATGVHSEINGSADHRMFSFHNGGVQISCKYPETV ICOS with of mouse ICOSQQLKMRLFREREVLCELTKTKGSGNAVSIKNPMLCLYHLSNNSVSFF signal(signal peptide is LNNPDSSQGSYYFCSLSIFDPPPFQERNLSGGYLHIYESQLCCQLKIpeptide underlined) VVQVTE 513 Cynomolgus Amino acid sequenceMKSGLWYFFL FCLHMKVLTG EINGSANYEM FIFHNGGVQI ICOS of cynomolgus ICOSLCKYPDIVQQ with (signal peptide isFKMQLLKGGQILCDLTKTKGSGNKVSIKSLKFCHSQLSNNSVSFFLY signal underlined) NLDpeptide RSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGC ATFVVVCIFGCILICWLTKKKYSSTVHDPNGEYMFMRAVNTAKKSRLTGT TP 514 CynomolgusAmino acid sequence EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTK ICOSof cynomolgus ICOS TKG extracellular extracellular domainSGNKVSIKSLKFCHSQLSNNSVSFFLYNLDRSHANYYFCNLSIFDPP domainPFK VTLTGGYLHIYESQLCCQLK 515 Human Amino acid sequenceDTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTY ICOSof human ICOS ligand HIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHligand comprising CLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTextracellular domain SINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKN AATWS 516 HumanAmino acid sequence MRLGSPGLLFLLFSSLRADTQEKEVRAHVGSDVELSCACPEGSRFDL ICOSof human ICOS ligand NDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGligand including signal DFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPpeptide VVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCL QHSYAGAWAVSPETELTGHVSEQ ID NO: 610 ICOSL-FcDTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSDIEGRMD PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLinker is underlined and in bold. Sequence preceding linker is human ICOSL (B7-H2).Sequence following linker is human IgG1 Fc. 517 C-terminal aminoAmino acids 21 to LQMILNGINNYKNPKLT A MLTFKFYMPKKATELKHLQCLEacid sequence of 133 of hIL-2 withEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE hIL-2 R38W mutationTTFMCEYADETATIVEFLNRWITFCQSIISTLT (bold & underlined) 518C-terminal amino Amino acids 21 to LQMILNGINNYKNPKLT QMLTFKFYMPKKATELKHLQCLE acid sequence of 133 of hIL-2 withEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE hIL-2 R38Q mutationTTFMCEYADETATIVEFLNRWITFCQSIISTLT (bold & underlined) 519 STIM002-Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCA Corrected Lightsequence of CCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCA chain variablecorrected V_(L) of GAGCCTCCTGCATAGTGATGGATACAACTATTTGGATTGG regionSTIM002 TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGAC CAAGCTGGAGATCAAA 520 STIM002-Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCA Corrected fullsequence of CCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCA light chaincorrected STIM002 GAGCCTCCTGCATAGTGATGGATACAACTATTTGGATTGG sequencelight chain TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctt tcaaccggggcgagtgt 521 STIM003-Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGC Corrected heavysequence of CTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGT chain variablecorrected V_(H) of CACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCT regionSTIM003 CCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTC ACCGTCTCCTCA 522 STIM003-Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGC Corrected fullsequence of CTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGT heavy chaincorrected STIM003 CACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCT sequenceheavy chain CCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCA AGTGATGA 523 Human IgG1 IGHGHuman Heavy Chain gcctccaccaagggcccatcggtcttccccctggcaccct constant 1*03Constant Region cctccaagagcacctctgggggcacagcggccctgggctg region(IGHG1*03) cctggtcaaggactacttccccgaaccggtgacggtgtcg Nucleotidetggaactcaggcgccctgaccagcggcgtgcacaccttcc Sequencecggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtccccgggtaaa524 Human Heavy Chain ASTKGPSVFPLAPSSKSTSG Constant RegionGTAALGCLVKDYFPEPVTVS (IGHG1*03) Protein WNSGALTSGVHTFPAVLQSS SequenceGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 525 Human IgG1 IGHG Human Heavy Chaingcctccaccaagggcccatcggtcttccccctggcaccct constant 1*04 Constant Regioncctccaagagcacctctgggggcacagcggccctgggctg region (IGHG1*04)cctggtcaaggactacttccccgaaccggtgacggtgtcg Nucleotidetggaactcaggcgccctgaccagcggcgtgcacaccttcc Sequencecggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacatcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtctccgggtaaa526 Human Heavy Chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Constant Region WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT(IGHG1*04) Protein YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG SequencePSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYT QKSLSLSPGK 527 Human IgG2 IGHGHuman Heavy Chain gcctccaccaagggcccatcggtcttccccctggcgccct constant 2*01Constant Region gctccaggagcacctccgagagcacagccgccctgggctg region &(IGHG2*01) cctggtcaaggactacttccccgaaccggtgacggtgtcg IGHG Nucleotidetggaactcaggcgctctgaccagcggcgtgcacaccttcc 2*03 Sequencecagctgtcctacagtcctcaggactctactccctcagcag &cgtggtgaccgtgccctccagcaacttcggcacccagacc IGHGtacacctgcaacgtagatcacaagcccagcaacaccaagg 2*05tggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggtaaa 528Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSConstant Region WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT(IGHG2*01) Protein YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF SequenceLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 529 Human IgG2 IGHGHuman Heavy Chain GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCT constant 2*02Constant Region GCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTG region(IGHG2*02) CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG NucleotideTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCC SequenceCGGCTGTCCTACAGTCCTCACGACTCTACTCCCTCAGCAGCGTGGTGACCGTGACCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTCCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCATGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTC TCCCTGTCTCCGGGTAAA 530Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSConstant Region WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQT(IGHG2*02) Protein YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF  SequenceLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 531 Human IgG2 IGHGHuman Heavy Chain gcctccaccaagggcccatcggtcttccccctggcgccct constant 2*04Constant Region gctccaggagcacctccgagagcacagcggccctgggctg region(IGHG2*04) cctggtcaaggactacttccccgaaccggtgacggtgtcg Nucleotidetggaactcaggcgctctgaccagcggcgtgcacaccttcc Sequencecagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggtaaa 532Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS Constant Region WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT(IGHG2*04) Protein YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF SequenceLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 533 Human IgG2 IGHGHuman Heavy Chain GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCT constant 2*06Constant Region GCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTG region(IGHG2*06) CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG NucleotideTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCC SequenceCGGCTGTCCTACAGTCCTCACGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTCCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGrGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGrTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGrGTGGrCAGCGTCCTCACCGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCArcrCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCTCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTC TCCCTGTCTCCGGGTAAA 534Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSConstant Region WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT(IGHG2*06) Protein YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF SequenceLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 535 Human Cλ IGLC CλLight Chain GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCAC constant 7*03Constant Region CCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGT region(IGLC7*03) GTGTCTCGTAAGTGACTTCAACCCGGGAGCCGTGACAGTG NucleotideGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGTGGGAGTGG SequenceAGACCACCAAACCCTCCAAACAAAGCAACAACAAGTATGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCT 536 Cλ Light ChainGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFNPGAVTV Constant RegionAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWK (IGLC7*03) AminoSHRSYSCRVTHEGSTVEKTVAPAECS Acid Sequence 537 Human WT IGHG WT human IgG1gcctccaccaagggcccatcggtcttccccctggcaccct IgG1 1*01 nucleotidecctccaagagcacctctgggggcacagcggccctgggctg constant & sequence #2cctggtcaaggactacttccccgaaccggtgacggtgtcg region IGHGtggaactcaggcgccctgaccagcggcgtgcacaccttcc 1*05cggctgtcctacagtcctcaggactctactccctcagcag (IgG1)cgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtctccgggtaaa538 Human Cλ IGLC Cλ Light ChainGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV constant 2*01 Constant RegionAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK region Amino AcidSHRSYSCQVTHEGSTVEKTVAPTECS Sequence #2- Encoded by nucleotidesequence version A & B

TABLE S3 SEQ ID NOS: 539-562 hIgG1 FIT-Ig bispecific 1a anti-ICOSAntibody A STIM003 anti-PD-L1 Antibody B 84G09 Sequence FIT-IgSEQ ID NO: DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #1 539 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FIT-Ig SEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 540EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKV FIT-IgSEQ ID NO: DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3 541 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEChIgG1 FIT-Ig bispecific 1b anti-ICOS Antibody A 84G09 anti-ICOSAntibody B STIM003 FIT-Ig SEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 542IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FIT-Ig SEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 543GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV FIT-IgSEQ ID NO: DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3 544 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EChIgG1 FIT-Ig bispecific 2a anti-ICOS Antibody A STIM001 anti-PD-L1Antibody B 1D05 FIT-Ig SEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 545LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 546EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KI FIT-IgSEQ ID NO: DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3 547 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEChIgG1 FIT-Ig bispecific 2b anti-PD-L1 Antibody A 1D05 anti-ICOSAntibody B STIM001 FIT-Ig SEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 548IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 549GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKI FIT-IgSEQ ID NO: DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3 550 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN EChIgG1 FIT-Ig bispecific 3a anti-ICOS Antibody A STIM003 anti-PD-L1Antibody B 1D05 FIT-Ig SEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 551LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 552EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKV FIT-IgSEQ ID NO: DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3 553 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEChIgG1 FIT-Ig bispecific 3b anti-PD-Ll Antibody A 1D05 anti-ICOSAntibody B STIM003 FIT-Ig SEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 554IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 555GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV FIT-IgSEQ ID NO: DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3 556 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EChIgG1 FIT-Ig bispecific 4a anti-ICOS Antibody A STIM001 anti-PD-L1Antibody B 84G09 FIT-Ig SEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 557LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 558EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKV FIT-IgSEQ ID NO: DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3 559 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEChIgG1 FIT-Ig bispecific 4b anti-PD-L1 Antibody A 84G09 anti-ICOSAntibody B STIM001 FIT-Ig SEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 560IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 561GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV FIT-IgSEQ ID NO: DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3 562 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

TABLE S4Sequences of antibody heavy chain variable regions obtained from additional clonesCDRs are defined according to IMGT. CLONE_ID VH_NUCLEOTIDE_SEQUENCEVH_AMINO_ACID_SEQ HCDR1 HCDR2 HCDR3 CL-61091CAGGTTCAACTGATGCAGTCTGGAACTGAGGTGAAGAAGCCTGGGG QVQLMQSGTEVKKPGASVGYTFTTYG ISAYSGDT ARSSGWPHHYGMDVCCTCAGTGAAGGTCTCCTGCAAGACTTCTGGTTACACCTTTACCAC KVSCKTSGYTFTTYGITW SEQ IDSEQ ID NO: SEQ ID NO: 567 CTATGGTATCACTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGVRQAPGQGLEWMGWISAY NO: 565 566TGGATGGGATGGATCAGCGCTTACAGTGGTGACACAGACTATGCAC SGDTDYAQKFQGRVTVTTAGAAGTTCCAGGGCAGAGTCACCGTGACAACAGACACATCCACGAA DTSTNTAYMELRSLKSDDCACAGCCTACATGGAGTTGAGGAGCCTGAAATCTGACGACACGGCC TAVYYCARSSGWPHHYGMGTGTATTATTGTGCGAGAAGTAGTGGCTGGCCCCACCACTACGGTA DVWGQGTTVTVSSTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG SEQ ID NO: 564 SEQ ID NO: 563CL-64536 CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAAAAGCCTGGGGQVQLVQSGGEVKKPGASV GYTFTSYG ISAYNGNT ARSTSYYGSGTLYGMDVCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAG KVSCKASGYTFTSYGFSW SEQ IDSEQ ID NO: SEQ ID NO: 570 CTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGVRQAPGQGLEWMGWISAY NO: 377 378TGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCAC NGNTNYAQKLQGRVSMTTAGAAGCTCCAGGGCAGAGTCTCCATGACCACAGACACATCCACGAG DTSTSTAYMELRSLRSDDCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCC TAVYFCARSTSYYGSGTLGTGTATTTCTGTGCGCGATCTACGTCTTACTATGGTTCGGGGACCC YGMDVWGQGTTVTVSSTATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC SEQ ID NO: 569 CTCAGSEQ ID NO: 568 CL-64837 CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGQVQLVQSGGEVKKPGASV GYTFTSYG ISAYNGNT ARSTSYYGSGTLYGMDVCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAG KVSCKASGYTFTSYGFSW SEQ IDSEQ ID NO: SEQ ID NO: 570 CTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGVRQAPGQGLEWMGWISAY NO: 377 378TGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCAC NGNTNYAQKLQGRVSMTTAGAAGCTCCAGGGCAGAGTCTCCATGACCACAGACACATCCACGAG DTSTSTAYMELRSLRSDDCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCC TAVYYCARSTSYYGSGTLGTGTATTACTGTGCGCGATCTACGTCTTACTATGGTTCGGGGACCC YGMDVWGQGTTVTVSSTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC SEQ ID NO: 572 CTCAGSEQ ID NO: 571 CL-64841 CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAAAAGCCTGGGGQVQLVQSGGEVKKPGASV GYTFTSYG ISAYNGNT ARSTSYYGSGTLYGMDVCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAG KVSCKASGYTFTSYGFSW SEQ IDSEQ ID NO: SEQ ID NO: 570 CTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGVRQAPGQGLEWMGWISAY NO: 377 378TGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCAC NGNTNYAQKLQGRVSMTTAGAAGCTCCAGGGCAGAGTCTCCATGACCACAGACACATCCACGAG DTSTSTAYMELRSLRSDDCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCC TAVYFCARSTSYYGSGTLGTGTATTTCTGTGCGCGATCTACGTCTTACTATGGTTCGGGGACCC YGMDVWGQGTTVTVSSTATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC SEQ ID NO: 574 CTCAGSEQ ID NO: 573 CL-64912 CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAAAAGCCTCGGGQVQLVQSGGEVKKPRASV GYTFTSYV ISGYNGNT ARSTSYYGAGTLYGMDVCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAG KVSCKASGYTFTSYVFSW SEQ IDSEQ ID NO: SEQ ID NO: 579 CTATGTGTTCAGCTGGGTGCGACATGCCGCTGGACAAGGACTAGAGVRHAAGQGLEWMGWISGY NO: 577 578TGGATGGGATGGATCAGCGGTTACAATGGTAACACAAACTATGCAC NGNTNYAQKLQCGVSMTAAGAAGCTCCAGTGCGGAGTCTCGATGACCGCAGACACATCCACGAG DTSTSTAYMELRSLRSDDCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCC TAVYFCARSTSYYGAGTLGTGTATTTCTGTGCGCGATCTACGTCTTACTATGGTGCGGGGACCC YGMDVWGQGTTVTVSSTATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC SEQ ID NO: 576 CTCAGSEQ ID NO: 575 CL-71642 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGEVQLVESGGGVVRPGGSL GFTFDDYG INWNGGST AADYYGSGSYYNVPFDYGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGA RLSCAASGFTFDDYGMSW SEQ IDSEQ ID NO: SEQ ID NO: 584 TTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGVRQAPGKGLEWVSGINWN NO: 582 583TGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTTATGCAG GGSTGYADSVKGRFTISRACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA DNAKNSLYLQMNSLRAEDCTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCC TALYYCAADYYGSGSYYNTTGTATTACTGTGCGGCCGATTACTATGGTTCGGGGAGTTATTATA VPFDYWGQGTLVTVSSACGTCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC SEQ ID NO: 581 CTCAGSEQ ID NO: 580 CL-74570 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGATACGGCCTGGGGEVQLVESGGGVIRPGGSL GFTFDDYG INWIGDNT ARDYFGSGSYYNVPFDYGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGA RLSCAASGFTFDDYGMSW SEQ IDSEQ ID NO: SEQ ID NO: 588 TTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGVRQAPGKGLEWVSGINWI NO: 582 587TGGGTCTCTGGTATTAATTGGATTGGTGATAACACAGATTATGCAG GDNTDYADSVKGRFTISRACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA DNAKNSLYLQMNSLRAEDCTCCCTATATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCC TALYYCARDYFGSGSYYNTTGTATTACTGTGCGAGAGATTACTTTGGTTCGGGGAGTTATTATA VPFDYWGQGTLVTVSSACGTTCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC SEQ ID NO: 586 CTCAGSEQ ID NO: 585

TABLE S5Sequences of antibody light chain variable regions obtained from additional clonesN terminal E and 5′nucleotide additions in CL-71642 are shown in bold. These were not recovered in sequencing but weredetermined to be present in the sequence by comparison against the related clones as shown in FIG 36. CDRs are definedaccording to IMGT. CLONE_ID VL_NUCLEOTIDE_SEQUENCE VL_AMINO_ACID_SEQLCDR1 LCDR2 LCDR3 CL-61091GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTG DIVMTQSPLSLPVTPGEPAQSLLHSNGFNY INS MQALQTPLT GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCASISCRSSQSLLHSNGFNYF SEQ ID NO: SEQ ID SEQ ID NO: 593TAGTAATGGATTCAACTATTTCGATTGGTACCTGCAGAAGCCAGGA DWYLQKPGQSPQLLIFLVS 591NO: CAGTCTCCACAGCTCCTGATCTTTTTGGTTTCTAATCGGGCCTCCG NRASGVPDRFSGSGSGTDF592 GGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTAC TLKISRVEAEDVGIYYCMQACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTAC ALQTPLTFGGGTKVEIKTGCATGCAAGCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCA SEQ ID NO: 590AGGTGGAGATCAAAC SEQ ID NO: 589 CL-64536GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTG DIVMTQSPLSLPVTPGEPAQSLLHSNGYNC IGS MQALQTPCS GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCASISCRSSQSLLHSNGYNCL SEQ ID NO: SEQ ID SEQ ID NO: 400TAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGG DWYLQKPGQSPQLLIYLGS 596NO: CAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCG TRASGFPDRFSGSGSGTDF371 GGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTAC TLKISRVEAEDVGVYYCMQACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTAC ALQTPCSFGQGTKLEIKTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCA SEQ ID NO: 595AGCTGGAGATCAAAC SEQ ID NO: 594 CL-64837GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTG DIVMTQSPLSLPVTPGEPAQSLLHSNGYNC IGS MQALQTPCS GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCASISCRSSQSLLHSNGYNCL SEQ ID NO: SEQ ID SEQ ID NO: 400TAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGG DWYLQKPGQSPQLLIYLGS 596NO: CAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCG TRASGFPDRFSGSGSGTDF371 GGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTAC TLKISRVEAEDVGVYYCMQACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTAC ALQTPCSFGQGTKLEIKTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCA SEQ ID NO: 598AGCTGGAGATCAAAC SEQ ID NO: 597 CL-64841GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTG DIVMTQSPLSLPVTPGEPAQSLLHSNGYNC IGS MQALQTPCS GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCASISCRSSQSLLHSNGYNCL SEQ ID NO: SEQ ID SEQ ID NO: 400TAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGG DWYLQKPGQSPQLLIYLGS 596NO: CAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCG TRASGFPDRFSGSGSGTDS371 GGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTCTAC TLKISRVEAEDVGVYYCMQACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTAC ALQTPCSFGQGTKLEIKTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCA SEQ ID NO: 600AGCTGGAGATCAAAC SEQ ID NO: 599 CL-64912GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTG DIVMTQSPLSLPVTPGEPAQSLLHSNGYNC IGS MQALQTPCS GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCASISCRSSQSLLHSNGYNCL SEQ ID NO: SEQ ID SEQ ID NO: 400TAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGG DWYLQKPGQSPQLLIYLGS 596NO: CAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCG TRASGFPDRFSGSGSGTDF371 GGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTAC TLKISRVEAEDVGVYYCMQACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTAC ALQTPCSFGQGTKLEIKTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCA SEQ ID NO: 602AGCTGGAGATCAAAC SEQ ID NO: 601 CL-71642GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG EIVLTQSPGTLSLSPGERAQSVSSSY GAS QQYGSSPFT GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTLSCRASQSVSSSYLAWYQ SEQ ID NO: SEQ ID SEQ ID NO: 605CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG QKPGQAPRLLIYGASSRAT 426NO: CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA GIPDRFSGSGSGTDFTLTI413 GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG SRLEPEDFAVYYCQQYGSSCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT PFTFGPGTKVDIKGGTAGCTCACCTTTCACTTTCGGCCCTGGGACCAAAGTGGATATCA SEQ ID NO: 604 AACSEQ ID NO: 603 CL-74570 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGEIVLTQSPGTLSLSPGERA QSVSSSY GAS HQYGNSPFTGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG TLSCRASQSVSSSYLAWYQSEQ ID NO: SEQ ID SEQ ID NO: 608CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG QKPGQAPRLLIYGASSRAT 426NO: CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA GIPDRFSGSGSGTDFTLTI413 GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG SRLEPEDFAVYYCHQYGNSCAGACTGGAACCTGAAGATTTTGCAGTATATTACTGTCACCAGTAT PFTFGPGTKVDIKGGTAATTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA SEQ ID NO: 607 AACSEQ ID NO: 606

What is claimed is:
 1. An isolated antibody that comprises a bindingsite for the extracellular domain of human ICOS, wherein the antibodycomprises a VH domain comprising an amino acid sequence having at least95% sequence identity to SEQ ID NO: 408 and a VL domain comprising anamino acid sequence having at least 95% sequence identity to SEQ ID NO:415.
 2. An antibody according to claim 1, wherein the VH domaincomprises a set of heavy chain complementarity determining regions(HCDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 has amino acid sequenceSEQ ID NO: 405, HCDR2 has amino acid sequence SEQ ID NO: 406, HCDR3 hasamino acid sequence SEQ ID NO:
 407. 3. An antibody according to claim 1,wherein the VL domain comprises a set of light chain complementaritydetermining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 hasamino acid sequence SEQ ID NO: 412, LCDR2 has amino acid sequence SEQ IDNO: 413, LCDR3 has amino acid sequence SEQ ID NO:
 414. 4. An antibodyaccording to claim 1, comprising a VH domain comprising amino acidsequence SEQ ID NO:
 408. 5. An antibody according to claim 1, comprisinga VL domain comprising amino acid sequence SEQ ID NO:
 415. 6. Anantibody according to claim 1 which is a human IgG.
 7. An antibodyaccording to claim 6 which comprises an effector enabled human IgG1constant region.
 8. An antibody according to claim 7, which is amultispecific antibody comprising a further antigen binding site.
 9. Anantibody according to claim 8, wherein the further antigen binding siterecognizes PD-L1.
 10. A composition comprising an antibody according toclaim 1 and an anti-PD-L1 antibody.
 11. A composition according to claim10, wherein the anti-PD-L1 antibody is a human IgG.
 12. A compositionaccording to claim 11, wherein the anti-PD-L1 antibody comprises aneffector enabled human IgG1 constant region.
 13. A composition accordingto claim 11, wherein the anti-PD-L1 antibody comprises a VH domainhaving amino acid sequence SEQ ID NO: 299 and a VL domain having aminoacid sequence SEQ ID NO:
 300. 14. A composition according to claim 13,wherein the anti-PD-L1 antibody is an immunocytokine comprising humanwild type or variant IL-2.
 15. A method of treating cancer in a patient,comprising administering an antibody according to claim 1 to thepatient.
 16. A method according to claim 15, wherein the cancer is renalcell cancer, head and neck cancer, melanoma, non small cell lung canceror diffuse large B-cell lymphoma.
 17. A method of treating cancer in apatient, comprising administering to the patient an antibody that bindsthe extracellular domain of human ICOS, wherein the antibody is a humanIgG1 comprising an effector enabled constant region, and an antibodythat binds human PD-L1, wherein the antibody is a human IgG1 comprisingan effector enabled constant region.
 18. A method according to claim 17,wherein the cancer is a renal cell cancer, head and neck cancer,melanoma, non small cell lung cancer or diffuse large B-cell lymphoma.19. A method according to claim 18, wherein the antibody that binds theextracellular domain of human ICOS and/or the antibody that binds humanPD-L1 is a human IgG1 comprising a wild type constant region.
 20. Amethod according to claim 19, wherein the constant region amino acidsequence is SEQ ID NO:
 340. 21. A method according to claim 17, whereinsaid antibody that binds the extracellular domain of human ICOS isadministered in a single dose, followed by administration of multipledoses of said antibody that binds human PD-L1.
 22. A method of treatingcancer in a patient, comprising administering to the patient an antibodythat binds the extracellular domain of human ICOS and which comprises aVH domain comprising an amino acid sequence having at least 95% sequenceidentity to SEQ ID NO: 408 and a VL domain comprising an amino acidsequence having at least 95% sequence identity to SEQ ID NO: 415,wherein said antibody is a human IgG1 comprising an effector enabledconstant region, and an antibody that binds human PD-L1, wherein saidantibody is a human IgG1 comprising an effector enabled constant region.23. A method according to claim 22, wherein said VH domain comprises aset of heavy chain complementarity determining regions (HCDRs) HCDR1,HCDR2 and HCDR3, wherein HCDR1 has amino acid sequence SEQ ID NO: 405,HCDR2 has amino acid sequence SEQ ID NO: 406, HCDR3 has amino acidsequence SEQ ID NO:
 407. 24. A method according to claim 22, whereinsaid VL domain comprises a set of light chain complementaritydetermining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 hasamino acid sequence SEQ ID NO: 412, LCDR2 has amino acid sequence SEQ IDNO: 413, LCDR3 has amino acid sequence SEQ ID NO:
 414. 25. A methodaccording to claim 23, wherein said VH domain comprises amino acidsequence SEQ ID NO:
 408. 26. A method according to claim 24, whereinsaid VL domain comprises amino acid sequence SEQ ID NO: 415.